Method and Apparatus for Analyzing Bioprocess Fluids

ABSTRACT

Methods and apparatus for analyzing bioprocess fluids are provided. A plurality of particles coated with a plurality of capture agents having an affinity for one or more biological markers is combined with bioprocess fluid to form a plurality of analyte-particle complexes. The system also includes a transport arrangement for transporting the sample to a sensor surface, and optionally a magnetic field inducing structure constructed and arranged to establish a magnetic field at and adjacent to the sensor surface. The resonant sensor produces a signal corresponding to an amount of analyte-particle complexes that are bound to the sensor surface.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/641,280, filed Dec. 19, 2006, which claims priority as acontinuation-in-part to each of U.S. application Ser. Nos. 11/416,789;11/417,000; 11/416,624; 11/417,005; 11/416,998; and 11/416,997 whichwere filed on May 2, 2006, now U.S. Pat. Nos. 7,648,844; 7,615,381;7,611,908; 7,598,094; 7,629,137; and 7,632,638, respectively, and whichclaim the benefit of and priority to U.S. Application No. 60/676,759filed May 2, 2005 and also claim priority as continuation-in-partapplications to U.S. application Ser. No. 11/183,484 filed Jul. 18,2005, now U.S. Pat. No. 7,300,631, which claims the benefit of andpriority to U.S. Application No. 60/690,592 filed Jun. 15, 2005, theteachings of all of which are incorporated herein in their entireties.U.S. patent application Ser. No. 11/641,280 also claims the benefit ofand priority to U.S. Application No. 60/751,492, filed Dec. 19, 2005,the teachings of which are incorporated herein in its entirety.

TECHNICAL FIELD

The present invention relates to methods for detecting one or morebiological markers in bioprocess fluid samples.

BACKGROUND OF THE INVENTION

Biological therapeutics such as proteins are produced in various animaland host cell models. Typically, the product molecule of interest isisolated from the host, host cell, or conditioned medium used to growthe host cell and is purified to remove host proteins and othercontaminants before being administered to humans. Sensitive measurementof such host proteins and contaminants is desirable at various pointsduring purification of the product molecule. Currently, techniques fordetecting or measuring the host cell proteins and contaminants duringpurification of product molecules typically involve Enzyme LinkedImmunosorbant Assay using antibodies that recognized the host cellprotein or contaminant. Such assays typically take at least 5 hours tocomplete and are typically performed off line, that is samples areremoved from the production line and taken to a separate laboratorywhere the assay is performed. The assays often have a detection limit ofabout 400 pg/ml.

Significant challenges for a system that detects analytes (e.g.,biological agents or biological markers) in liquid media includeconcentration of the analyte in the media, and transport of the analyteto a sensor surface. For biological applications such as analysis ofbioprocess fluids for host cell proteins or contaminants, concentrationissues generally arise since the concentrations of such analytes tend tobe low. Additionally, biological analytes (e.g., cells, cell fragmentsand macromolecules such as proteins and nucleic acids) tend to berelatively large; hence, transport issues arise because these largeranalytes diffuse in fluid solution very slowly. In addition to cells,cell fragments, and molecules such as proteins and nucleic acids, thedetection of small molecule analytes can be a useful marker fordiagnosing disease, monitoring drug pharmacokinetics in a patient,screening small molecule libraries for potential drug targets andanalyzing bioprocess fluids.

There is a need for improved assays that can quickly detect lowconcentrations of analyte. In addition, there is a need for improvedmeasurement of analytes including small molecule analytes. Furthermore,a need exists for highly sensitive methods and apparatus for analyzingbioprocess fluids and methods and apparatus that can be used in realtime at the production line, or even in line with production in order tostreamline the purification process. The ability to analyze bioprocessfluids at line or in line at various stages of the purification processis expected to allow more rapid troubleshooting of production problems,thereby minimizing production downtime.

A key metric for competitive detection is the amount of analyteaccumulated on a sensor per unit time. For good performance, the rate ofaccumulation (and the resulting signal transient) needs to be fastrelative to the sensor drift rate. Another key performance metric for ananalyte detection system is the degree to which the system canpreferentially collect the analyte of interest on the sensor surface.Since many biological samples contain extraneous background components(e.g., other proteins, cells, nucleic acids, dirt), it is necessary toprevent these background components from interfering with the desiredmeasurement. So, a transport method that selectively draws the analyteto the sensor and allows interfering background components to pass byhas definite advantages. Such a method used in concert with selectivebinding of the analyte (e.g., antibody, complimentary DNA strands, etc.)to the sensor surface can deliver high sensitivity measurements forsamples with large amounts of extraneous background components relativeto the amount of analyte.

Various methods for improving transport of analyte to a sensor surfacehave been proposed, including filtration, novel flow geometries,acoustic fields, electrical fields (time varying and static) andmagnetic fields.

Acoustic excitation has been used to draw cells to field nodes, but itis difficult to use this technique alone to transport material to asurface.

Electrical fields (electrophoresis and dielectrophoresis) have been usedto enhance transport but are not universally applicable to all analytesand sample types. They are generally more effective for larger analytes(e.g., cells). Furthermore, the electrical properties of microbes canvary within a given species and strain, making it hard to predict systemperformance under all intended operating conditions. Sometimes it isnecessary to tailor the ionic strength of the sample to improve theperformance of the transport. This requirement can conflict with theoptimum binding or wash conditions in an assay. Also, electrical fieldscan dissipate energy and heat conductive fluids (e.g., 0.1 M phosphatebuffer solution), which is undesirable since heating can damage thebiological analytes.

Immunomagnetic separation (IMS) methods are known in the art forisolating analyte from a sample.

SUMMARY OF THE INVENTION

In one embodiment, methods for analyzing a bioprocess fluid areprovided. In some embodiments, the method comprises introducing abioprocess fluid and a plurality of particles coated with a plurality ofcapture agents capable of binding one or more biological markers into afluid chamber. At least one surface of the fluid chamber comprises anacoustic device having a plurality of capture agents capable of bindingthe one or more biological markers bound thereto. Signal output by saidacoustic device is monitored, thereby detecting one or more biologicalmarkers in the bioprocess fluid.

In some embodiments, the analysis includes assessing the purity of abioprocess fluid. For example, in some embodiments, the method involvesdetecting a level of one or more biological markers in the bioprocessfluid. The method comprises introducing the bioprocess fluid and aplurality of particles coated with a plurality of capture agents capableof binding the biological marker into a fluid chamber. At least onesurface of the fluid chamber comprises an acoustic device having aplurality of capture agent capable of binding the one or more biologicalmarkers bound thereto. Signal output by said acoustic device ismonitored to detect the level of one or more biological markers in thesample, thereby assessing purity of a bioprocess fluid.

In some embodiments the method involves detecting a level of one or morebiological markers in the bioprocess fluid. The bioprocess fluid iscombined with a plurality of magnetic particles that comprise aplurality of capture agents having an affinity for one or morebiological markers to produce at least some magnetic particles bound toat least some of the one or more biological markers. The combined fluidis directed into a fluid chamber, wherein at least one surface of thefluid chamber comprises an acoustic device having a plurality of captureagents capable of binding the one or more biological markers boundthereto. A magnetic flux is created in proximity to the flexural platewave device to magnetically attract at least some of the plurality ofbound magnetic particles to the at least one surface of the flexuralplate wave device, and signal output by said acoustic device ismonitored, to detect the level of one or more biological markers in thesample, thereby assessing purity of a bioprocess fluid.

Apparatus for assessing purity of a bioprocess fluid is also provided.In some embodiments, the apparatus comprises a fluid chamber having atleast one opening for fluid to enter; a flexural plate wave devicedefining at least a portion of at least one interior surface of thefluid chamber. In some embodiments, the at least one interior surface iscapable of being coated with a plurality of capture agents that arecapable of binding one or more biological markers. In other embodiments,the surface is coated with a plurality of capture agents that arecapable of binding one more biological markers. The apparatus includesan monitoring device to monitor at least one signal output by theflexural plate wave device. In some embodiments, the apparatus includesa plurality of magnetic particles capable of being coated with aplurality of capture agents capable of binding one or more biologicalmarkers. In other embodiments, the plurality of magnetic particles arecoated with a plurality of capture agents that are capable of bindingone or more biological markers. The apparatus can also include a sourceof magnetic flux to selectively attract magnetic particles to theflexural plate wave device.

In one embodiment the biological marker binds to a magnetic particle(e.g., a magnetic bead) to form an analyte-particle complex. Theanalyte-particle complex is transported and localized onto the surfaceof a sensing device by applying a gradient magnetic field. The magneticfield induces a polarization in the magnetic material of the particlethat is aligned with the local magnetic field lines. The particleexperiences a net force in the direction of the gradient, causing theparticle to migrate toward regions of higher field strength. Themagnetic field distribution is tailored to draw analyte-particlecomplexes from a sample flow and distribute them across the surface ofthe sensing device. The extraneous, background components of the sample(e.g., cells, proteins) generally have a much lower magneticsusceptibility as compared to the magnetic particles, and so themagnetic field does not significantly influence them. Hence, only a verysmall fraction of this background material interacts with the sensorsurface.

In one embodiment, the sensing device is a flexural plate wave (FPW)device, which functions particularly well with the magnetic particlesfor two reasons. First, the presence of the magnetic particles on thesurface of the sensing device results in an amplified FPW signalresponse. The larger combined size and density of the analyte-particlecomplex yields a larger FPW signal response than the analyte alone.Second, the surface of the sensor in the FPW device consists of a thinmembrane that is typically only a few micrometers thick, which allowslarger magnetic fields and field gradients to be created at the sensorsurface because the field source can be positioned closer to the sampleflow. This results in higher fractional capture of the analyte from thesample. With this higher capture rate and efficiency, it is possible toprocess larger sample volumes in shorter times than would be otherwisepossible.

In another aspect, a cartridge for a resonant device system includes afirst fluid chamber having at least one opening for fluid to enter, anda flexural plate wave device defining at least one interior surface ofthe fluid chamber. The apparatus further includes a first source ofmagnetic flux to selectively attract magnetic particles to the at leastone interior surface of the first fluid chamber.

In another aspect, a method for detection of a biological marker (alsoreferred to herein as an analyte) includes combining a fluid containingan analyte (also referred to herein as bioprocess fluid) with aplurality of magnetic particles that comprise a capture agent having anaffinity for the analyte to produce at least some magnetic particlesbound to at least some analyte. The method further includes directingthe combined fluid into a first fluid chamber, wherein at least onesurface of a flexural plate wave device is in fluid communication withthe fluid in the first fluid chamber. The method also includes creatinga first magnetic flux in proximity to the flexural plate wave device tomagnetically attract at least some of the bound magnetic particles tothe at least one surface of the flexural plate wave device.

In another aspect, a method for detection of an analyte includes coatingat least a portion of a surface of a flexural plate wave device locatedin a fluid chamber with a first capture agent, and directing a fluidcontaining an analyte into the fluid chamber to bind some of the analyteto the capture agent located on the flexural plate wave device. Themethod further includes directing a fluid containing a plurality ofmagnetic particles that comprise a second capture agent into the fluidchamber, and creating a magnetic flux in proximity to the flexural platewave device to attract at least some of the magnetic particles towardsthe surface of the flexural plate wave device.

The methods and apparatus provided herein can be used at any stageduring the processing and/or purification of the product molecule ofinterest. Typically, processing and/or purification of product moleculesinvolves the stages of feed preparation, capture of the productmolecule, intermediate purification and polishing. A sample of thebioprocess fluid can be tested using the method and or apparatusprovided herein before, during, or after any one of the stages. Thebioprocess fluid can be tested for the presence of one or morebiological markers. The bioprocess fluid can also be tested for thepresence and/or amount of the product molecule itself. In addition, theflow through liquid of any relevant purification step can be tested forthe presence and/or amount of product molecule. In this manner, thepurity of the bioprocess fluid and the yield of the product molecule ofinterest can be monitored.

For example, in many bioprocessing applications, the initial sample orfeed stream often has properties that interfere with or reduce theefficacy of downstream purification or processing methods. The sample orfeed stream may have, for example, biological markers such as nucleicacids, endotoxins, cell debris, and/or particulates. Techniques such asfiltration can be used to remove the biological markers while retainingthe product molecule of interest in the bioprocessing fluid. Dependingon the size and concentration of the biological marker and productmolecule of interest, micro-filtration, ultra filtration, ordiafiltration or combinations thereof can be used. The resultingbioprocess fluid can be analyzed for the presence and/or amount of thebiological markers as well as for the presence and/or amount of theproduct molecule.

After conditioning the initial sample, the product molecule is recoveredand/or stabilized from the relatively crude feedstock. The recoveryprocess typically involves some form of binding process, where theproduct molecule is reversibly bound to a solid support, while the restof the feedstock is washed away. Reversible binding of the productmolecule can be achieved, for example, using one or more antibodies thatspecifically bind the product molecule, ion exchange chromatography(IEC), or hydrophobic interactions. Typically biological markers thathave very different pI, hydrophobicity, size, and/or antigenicproperties from the product molecule are removed. Further, anybiological markers that can damage the product molecule, such asproteases are also removed, while recovering a maximal amount of theproduct molecule. The resulting bioprocess fluid can be analyzed for thepresence and/or level of the biological markers as well as the presenceand/or level of the product molecule. In addition, the flow throughliquid from the capture stage can be tested for the presence and/orlevel of product molecule that was not captured, thereby allowing theoperator to optimize the efficiency of the capture process at line or inline with the process.

After the capture stage, intermediate purification can be performed.After intermediate purification, steps can be taken to remove anyremaining trace contaminants or biological markers that have beendifficult to remove due to the similarities with the product moleculecan be removed from the bioprocess fluid. Techniques such as IEC,hydrophobic interaction chromatography, and/or gel filtration are oftenused for intermediate purification and polishing with more selectivewashing or elution buffers in order to remove biological markers thatare more similar in size, charge, and/or hydrophobicity to the productmolecule of interest. Like the capture stage, the flow through liquidand resulting bioprocess fluid can be analyzed for the presence and/oramount of product molecule as well as the presence and/or amount ofbiological markers.

As a result of the methods and apparatus described herein, it is nowpossible to analyze a bioprocess fluid for the presence and/or amount ofproduct molecule as well as the presence and/or amount of biologicalmarkers such as contaminiants on the same platform. For example, onesurface of the apparatus can be coated with a capture agent that bindsthe product molecule and another surface coated with a capture agentthat binds one or more biological markers. The sample can be introducedto the first and second surfaces and the presence and/or amount ofproduct molecule can be measured by monitoring the frequency shift ofthe first surface. The sample can be introduced to the second surface inconjuction with particles (also referred to herein as beads), that havebeen coated with capture agent as described supra. The presence and/oramount of the biological marker can be measuered using the secondsurface.

Where the product molecule is present at concentrations of 1 mg/ml ormore, the sample of bioprocess fluid can be diluted as necessary beforeintroducing the sample to the functionalized surface. In someembodiments, the bioprocess fluid is diluted by a factor of about 10. Inother embodiments, the bioprocess fluid is diluted by a factor of about100.

In some embodiments, the functionalized surface can be calibrated usingknown concentrations of analytes. In some embodiments, thefunctionalized surface can be reused. After use, analyte and/or analytebound to particles can be removed by washing the surface with a suitablewashing solution. In some embodiments, the washing solution can bephosphoric acid. The washing solution can be, for example, 0.1Mphosphoric acid. The washing solution can be flowed over the surface fora suitable period of time to remove the analyte and/or analyte/particlecomplexes. In some embodiments, the washing solution is flowed over thesurface for 1 minute. In this manner, the functionalized surfaces can bereused multiple times. In some embodiments, the surfaces can be reused10 or more times. A calibrated functionalized surface need not berecalibrated after each use or after stripping away the analyte oranalyte/particle complexes.

As a result of the present invention, lower levels of analyte can bedetected compared to conventional assays. The methods and apparatusprovided herein allow for analysis of the bioprocess fluid in less timeand can be performed at the production line.

Surprisingly, the use of fewer particles per sample allows for moresensitive detection of analyte. This was found where the size of theanalyte is on the order of the size of the particle, and where theanalyte is a small molecule. Furthermore, devices fabricated and methodsperformed according to the principles of the present invention areparticularly useful for capturing low concentrations of particlesbecause the sensor surface is thin (for example, in one embodiment, onthe order of several microns). In one embodiment, the thin surface isused in conjunction with a magnetic field gradient and magneticparticles. Because the sensor surface is thin, a large magnetic fieldgradient can be induced one the side of the sensor surface. The largemagnetic field gradient enhances the attraction of the magneticparticles to the sensor surface by focusing the field close to thesurface of the sensor, while allowing other material contained in thesample to flow by. In this way, lower numbers of particles can be used,thereby improving the detection limit of the system, because themagnetic field gradient serves to concentrate the particles near thesensing surface where they can interact with the bound analyte orcapture agent, depending on the assay format. The magnetic fieldgradient is removed to allow any non-specifically bound particles to bewashed away. Thus, the present invention improves the accuracy and thesensitivity of analyte detection.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing and other objects of this invention, the various featuresthereof, as well as the invention itself, may be more fully understoodfrom the following description, when read together with the accompanyingdrawings in which:

FIG. 1A shows one embodiment of an analyte detection system, constructedaccording to the invention.

FIG. 1B shows another embodiment of a portion of the analyte detectionsystem shown in FIG. 1A.

FIG. 2A shows a more detailed view of the FPW sensor shown in FIG. 2.

FIG. 3 shows a general detection protocol for using an FPW sensor incombination with analyte-particle complexes for detection of biologicalanalytes.

FIG. 4 shows the change in the signal from multiple FPW sensors as afunction of time for an exemplary detection protocol.

FIG. 5 is a summary plot showing the final signal change detected as afunction of original analyte concentration.

FIG. 6 shows a time evolution plot for a detection protocol, similar tothe plot shown in FIG. 4, but rather for a PSA analyte.

FIG. 7 is a schematic of two formats for attaching a capture agent tothe sensing surface of the acoustic device.

FIG. 8 is a schematic of one embodiment of the present invention, wherecompetitor molecule (e.g., analyte) is bound to the sensing surface.

FIG. 9 is a schematic of one embodiment of the present invention, wherethe competitor molecule is the analyte of interest linked to a tag andthe sensing surface of the acoustic device is coated with a captureagent that is capable of binding to the tag.

FIG. 10 is a schematic of one embodiment of the present invention, wherethe competitor molecule comprises a carrier and two or more analytemolecules of interest bound thereto, and the sensing surface of theacoustic device is coated with a capture agent that is capable ofbinding to the analyte of interest.

FIG. 11 shows the change in signal from multiple FPW sensors as afunction of PSA concentration.

FIG. 12 shows the change in signal from multiple FPW sensors as afunction of estradiol concentration in serum.

FIG. 13 is a dose response curve of FK-506 in buffer.

FIG. 14 shows the change in signal from multiple FPW sensors as afunction of FK-506 concentration in buffer.

FIG. 15 is a dose response curve of FK-506 in serum.

FIG. 16 shows the change in signal from multiple FPW sensors as afunction of FK-506 concentration in serum.

FIG. 17 shows the change in signal from multiple FPW sensors as afunction of particle concentration.

FIG. 18 shows a dose response curve of c-Troponin-I in buffer.

FIG. 19 shows a dose response curve of c-Troponin-I in serum.

FIG. 20 shows a dose response curve of c-Troponin-I in lysed wholeblood.

FIG. 21 shows a dose response curve of CHO-HSP in buffer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is drawn to methods of detecting one or morebiological markers (also referred to herein as an analyte) in a sample,such as a bioprocess fluid, using an acoustic device. In general terms,an analyte is detected based on the ability of particles coated with acapture agent (also referred to herein as a first capture agent) toalter the resonating frequency of an acoustic device in the presence ofthe analyte. The capture agent is capable of binding to the analyte. Aplurality of particles coated with a capture agent is exposed to thesensing surface of the acoustic device in the presence of the sample,and optionally in the presence of a competitor molecule. Depending onthe type of analyte to be detected, the sensing surface is coated with acapture agent (also referred to herein as a second capture agent)capable of binding the analyte (e.g., a sandwich binding format), or thesensing surface is coated with a competitor molecule (e.g., a smallmolecule format). The sensing surface is part of a fluid chamber orchannel as described herein. In addition, the coated particles and/orsample can be exposed to the sensing surface in a static mode, forexample, the coated particles and/or sample can be introduced into thechamber and incubated with the period for a given period of time. Inanother embodiment, the coated particles can be exposed to a sensingsurface in a non-static mode, for example, by flowing the coatedparticles through the fluid chamber or channel.

Generally, in the sandwich assay formats of the present invention, theamount of particles capable of binding to the sensing surface of theacoustic device is proportional to the amount of analyte present in thesample. Higher levels of analyte in the sample results in more of thecapture agent on the particles being occupied by analyte from thesample. Analyte coated particles are then able to bind to the captureagent coated sensing surface. In a preferred embodiment, the particlesare magnetic, and a magnetic flux is applied in proximity to theacoustic device to attract at least one of the plurality of magneticparticles toward the sensing surface.

As described herein, it was surprisingly found that using a lowerconcentration of particles allowed for the detection of lowerconcentrations of analytes. The finding was unexpected becauseconventional wisdom in assays involving capture by particles calls forthe use of a high quantity of particles in order to maximize captureefficiency. However, as shown in FIG. 17, it was found that detectionlimits were greatly affected by particle concentration and that use oflower particle concentrations allowed for the detection of analytes atlower concentration. Briefly, various concentrations of live E. coliwere incubated with 3×10⁵, 3×10⁴, or 6×10³ dynal beads and analyzed bythe method of the present invention.

Lower particle concentration also lowers capture efficiency. However, onbalance, the large improvement in detection limit outweighed the smallereffect on capture efficiency. While not wishing to be bound by theory,in the case of an analyte of a size on the order of the particle, thebinding of one analyte per particle is thought to be enough to allow theanalyte-bound particle to bind the capture agent coated surface, in partbecause the bound analyte typically contains multiple sites that can bebound by the capture agent. Use of a large number or high concentrationof particles results in many particles that have no analyte bound, whichthen increases the background signal, by increasing the amount ofparticles binding the surface non-specifically. Furthermore, if theconcentration of particles is high, the non-specifically bound particlesmay block or prevent particles that have bound analyte from binding tothe surface. The particles can be used in a concentration from about1×10² to about 1×10⁷. In another embodiment, the particles are at aconcentration of about 5×10³ to about 5×10⁵.

FIG. 7 shows two embodiments where the sensing surface 12 of theacoustic device is coated with a capture agent. In FIG. 7A, the captureagent 14 is bound directly to the sensing surface 12. In FIG. 7B, thecapture agent 14 is labeled with a first member of a binding pair 32.The surface is coated with the first member of a binding pair 32 and thesecond member of the binding pair 22.

As shown in FIG. 8, in one embodiment of the small molecule format, thecompetitor molecule 24 comprises the analyte of interest 10 bound to thesensing surface 12 of the acoustic device (FIG. 8A). Particles 14 coatedwith capture agent 16 are exposed to sample containing the analyte 10(FIG. 8B). Particle-bound capture agent that that has not bound analytefrom the sample 20 is free to bind the analyte bound to the surface ofthe acoustic device (FIG. 8C). As shown in FIGS. 8C and 8D, higherlevels of analyte in the sample result in fewer particles binding theacoustic device sensing surface because more of the capture agentpresent on the particles is occupied with analyte from the sample.Signal output by the acoustic device is monitored to determine theamount and/or number of particles that have bound to the surface,thereby detecting whether one or more analytes is present in a sample.In addition, the presence or amount of analyte present in the sample canbe determined, for example by comparing the signal to a control. In analternative embodiment, as described herein, the particles can bemagnetic particles. After introducing the particles into the fluidchamber, magnetic flux is created in proximity to the acoustic device toattract at least one of the plurality of magnetic particles toward thesensing surface (similarly as described, for example, in FIGS. 1A, 1Band 2).

In another embodiment of the small molecule format, as shown in FIG. 9,the sensing surface 12 of the acoustic device is coated with a captureagent 22 (also referred to herein as a second capture agent) that iscapable of binding to the competitor molecule 24. The competitormolecule can be, for example, the analyte of interest 10 bound to a tag26, and the second capture agent is capable of binding to the tag. Asshown in FIG. 9, higher levels of analyte in the sample results in fewerparticles 14 binding to the sensing surface of the acoustic devicebecause more of the capture agent present on the particles 16 isoccupied with analyte from the sample. Because the second capture agentis capable of binding to the tag portion of the competitor molecule, thecoated particles bind to the sensing surface of the acoustic device onlyif the particles have bound the competitor molecule.

In another embodiment of the small molecule format, as shown in FIG. 10,the competitor molecule 24 can be, for example, two or more analytemolecules or moieties of interest 10 bound to a carrier 28 and thesecond capture agent 40 is capable of binding the analyte. As shown inFIG. 10A, the second capture agent can be indirectly bound to theacoustic device sensing surface. The surface-bound and particle-boundcapture agents can be the same capture agent. Particles 14 coated withcapture agent 16 are exposed to sample containing the analyte 10 and thecompetitor molecule 24 (FIG. 10B). Particle-bound capture agent thatthat has not bound analyte from the sample is free to bind thecompetitor molecule, which in turn is bound by the capture agent that isbound to the sensing surface (FIG. 10C). As shown in FIG. 10D, higherlevels of analyte present in the sample results in fewer particlesbinding the sensing surface of the acoustic device because more of thecapture agent present on the particles is occupied with analyte from thesample. Where the analyte is a small molecule having only one copy of agiven epitope or binding site, the coated particles will bind to theacoustic device sensing surface only if the particles have bound to thecompetitor molecule.

The method of the present invention can be used to determine theconcentration or level of the analyte in the sample. The concentrationdetected or measured by the method of the present invention can be anabsolute concentration or a relative concentration. For example, theconcentration may be a ratio relative to the concentration of areference analyte present in the same sample of body fluid. Theconcentration can be obtained by comparing the data with similar dataobtained at a different time to determine whether a significant changein actual concentration has occurred. In addition, the present inventioncan be used in a binary format that provides a read-out that is positiveif the amount of analyte detected is above a predetermined level orconcentration and/or negative if the amount of analyte detected is belowa predetermined level or concentration. In other embodiments, the methodcan provide a positive read-out if a certain threshold level of analyteis detected.

For each of the embodiments described herein, a number of variations canbe made to the method as described below, without departing from thescope of the invention.

Samples

Samples suitable for use in the present invention includes any materialsuspected of containing the analyte. The sample can be used directly asobtained from the source or following one or more steps to modify thesample. In one embodiment, the sample is a biological sample. The samplecan be derived from any biological source, such as a physiologicalfluids (e.g., blood, saliva, sputum, plasma, serum, ocular lens fluid,cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid,peritoneal fluid, amniotic fluid, and the like) and fecal matter. Thesample can be obtained from biological swabs such as nasal or rectalswabs. In addition, the sample can be biopsy material. The sample can beobtained from a human, primate, animal, avian or other suitable source.In some embodiments, the sample is a bioprocess fluid. The bioprocessfluid can contain, for example, a product molecule that is to bepurified from the bioprocess fluid. The bioprocess can be any samplethat contains or is expected to contain the product molecule. Forexample, the bioprocess fluid can be conditioned tissue culture medium,milk, blood, plasma, plasma fractions, ground tissue, urine, ascitesfluid and the like.

The bioprocess fluid can contain recombinantly produced productmolecule. In some embodiments, the bioprocess fluid is conditionedmedium produced by a recombinant host cell. As described herein, therecombinant host cell can be any suitable cell that has been altered togenerate the product molecule, for example, by expressing an exogenousgene or nucleic acid that codes for the product molecule. In someembodiments, the recombinant host cell is selected from the groupconsisting of bacteria, fungi, insect cells, and mammalian cells intowhich one or more exogenous nucleic acid sequences have been added. Suchnucleic acid sequences include constructs that are capable ofexpressing, for example, a protein of interest. The exogenous nucleicacid sequence can be introduced into the host cell by, for example,transfection using suitable transfection methods known in the art, orcan be introduced by infection using a suitable viral vector, such thatthe protein encoded by the exogenous nucleic acid is expressed in thehost cell. Suitable bacteria include, for example, E. coli. Suitablefungi include, for example, Saccharomyces cerevisiae or Pichia pastoris.Suitable insect cells include, for example, SF9 cells. Recombinant hostcells include mammalian cells. Suitable recombinant mammalian cellsinclude, for example, hybridoma cells or transfected mammalian cells.Suitable transfected mammalian cells include, for example, SP2/0 cells,NS/0 cells, Chinese Hamster Ovary (CHO) cells, or Human Embryonic Kidney(HEK) 293 cells to which exogenous nucleic acid has been added asdescribed above.

In other embodiments, the bioprocess fluid can be one or more liquidsselected from the group consisting of milk, conditioned cell culturemedia, blood, serum, plasma, and plasma fractions. In other embodiments,the bioprocess fluid comprises one or more liquids selected from thegroup consisting of cell homogenates and tissue homogenates.

In some embodiments, the bioprocess fluid can contain an endogenousproduct molecule, for example, clotting factors and immunoglobulin canbe isolated from blood or fractions thereof.

As described above, the methods and apparatus provided herein can beused to analyze bioprocess fluids at all stages of the process used topurify the product from the starting material. As used herein,bioprocess fluid includes the sample as initially obtained (asconditioned cell culture medium, blood, plasma, plasma fractions, milk,tissue homogenates and the like), or can be the product of one or moreprocessing steps to which the sample is subjected during the procedureused to isolate or purify the product from the sample. In oneembodiment, the bioprocess fluid has been subjected to one or morepurification steps. Such purification steps include, for example,centrifugation, filtration, diafiltration, size fractionation, affinitychromatography, IEC, HIC, gel filtration, dilution, and the like.

In some embodiments, the purified product molecule contains no more thanabout 1-10 ng/ml of host cell protein, such as CHO—HCP, no more thanabout 1 ng/ml of endotoxin, no more than about 0.1 ng/ml of DNA, no morethan about 1% of aggregates of the product molecule, and sub milligramquantities of protein A.

As described below, the sample can be pretreated prior to use, such aspreparing plasma from blood, diluting viscous fluids, and the like. Thesample can also be filtered, distilled, extracted, digested with enzymeor concentrated. In one embodiment, a blood sample is obtained from anindividual and centrifuged and the plasma is analyzed. The sample canalso be treated to inactivate or modify certain activities in the samplecapable of interfering with the analyte or the detection process. Forexample, a decomplexing antagonist can be added to the sample todisassociate the analyte from other molecules that may be bound toand/or may interfere with the ability of the capture agent to bind tothe analyte. Such antagonists can be, for example, steroid antagonists.In the case of estradiol detection, the sample can be treated by addingdanazol to disassociate estradiol from sex hormone binding protein.

Other samples besides physiological fluids and solids can be used, suchas water, food products, and the like, for the performance ofenvironmental or food production assays. For example, the sample can bemeat, or poultry wash (the solution used to wash poultry). In addition,a solid material suspected of containing the analyte can be used as thetest sample. A solid test sample can be modified (e.g., homogenized,extracted, stomached, or solubilized) to form a liquid medium or torelease the analyte.

The sample volume can be as little as 10 μl or as much as 250 ml. Inanother embodiment, the sample volume is about 1 to about 5 ml.

Capture Agents

Suitable capture agents for use in the present invention include anymolecule capable of binding to one or more analytes (e.g., biologicalmarkers or product molecule) of interest. The term “capture agent”includes molecules or multi-molecular complexes that can bind to ananalyte or biological marker of interest. Capture agents preferably bindto their binding partners in a substantially specific manner. Captureagents with dissociation constants (KD) of less than about 10⁻⁶ arepreferred. The capture agent can also be, for example, polypeptides,nucleic acids, carbohydrates, nucleoproteins, glycoproteins, glycolipidsand lipoproteins.

In some embodiments, it is desirable to detect the presence or amount ofproduct molecule in the bioprocess fluid. Capture agents includesinclude molecules that can bind to the product molecule. In someembodiments, the capture agent is protein A, protein G, or othermolecule capable of binding to immunoglobulin.

Antibodies or antibody fragments are highly suitable as capture agents.Antigens may also serve as capture agents, since they are capable ofbinding antibodies. A receptor which binds a ligand is another exampleof a possible capture agent. Protein-capture agents are understood notto be limited to agents which only interact with their binding partnersthrough noncovalent interactions. Capture agents may also optionallybecome covalently attached to the proteins which they bind. For example,the capture agent may be photocrosslinked to its binding partnerfollowing binding.

As used herein, “plurality of capture agents” includes a mixture ofdifferent capture agents such that two or more proteins or two or moreantigens that comprise the one or more biological markers can be boundby the plurality of capture agents.

The term “antibody” includes any immunoglobulin, whether naturallyproduced or synthetically produced in whole, or in part. Derivatives ofantibodies that maintain the ability of the antibody to bind to theanalyte of interest are also included in the term. The term alsoincludes any protein having a binding domain which is homologous orlargely homologous to an immunoglobulin binding domain. These proteinsmay be derived from natural sources, produced synthetically in whole orin part. In one embodiment, the plurality of capture agents comprises apolyclonal antibody. In another embodiment, the plurality of captureagents comprises a combination of two or more monoclonal antibodies. Theantibody may be a member of any immunoglobulin class, including IgG,IgM, IgA, IgD, and IgE. Where the analyte is known to bind a carrierprotein, the antibody can be specific for the free form of the analyteor the carrier-bound form of the analyte. Antibodies that are capable ofbinding an analyte of choice can be obtained commercially or producedusing known methods for generating antibodies.

The term antibody also includes antibody fragments. The term “antibodyfragments” refers to any derivative of an antibody which is less thanfull-length. Preferably, the antibody fragment retains at least theability to bind the analyte of interest. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv, dsFvdiabody, and Fd fragments. The antibody fragment may be produced by anymeans. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody or it may berecombinantly produced from a gene encoding the partial antibodysequence. Alternatively, the antibody fragment may be syntheticallyproduced in whole or in part. The antibody fragment may optionally be asingle chain antibody fragment. Alternatively, the fragment may comprisemultiple chains which are linked together, for instance, by disulfidelinkages. The fragment may also optionally be a multimolecular complex.A functional antibody fragment will typically comprise at least about 50amino acids and more typically will comprise at least about 200 aminoacids.

Single-chain Fvs (scFvs) are recombinant antibody fragments consistingof only the variable light chain (V_(L)) and variable heavy chain(V_(H)) covalently connected to one another by a polypeptide linker.Either V_(L) or V_(H) may be the NH₂-terminal domain. The polypeptidelinker may be of variable length and composition so long as the twovariable domains are bridged without serious steric interference.Typically, the linkers are comprised primarily of stretches of glycineand serine residues with some glutamic acid or lysine residuesinterspersed for solubility. “Diabodies” are dimeric scFvs. Thecomponents of diabodies typically have shorter peptide linkers than mostscFvs and they show a preference for associating as dimers. An “Fv”fragment is an antibody fragment which consists of one V_(H) and oneV_(L) domain held together by noncovalent interactions. The term “dsFv”is used herein to refer to an Fv with an engineered intermoleculardisulfide bond to stabilize the V_(H)-V_(L) pair. A “F(ab′)₂” fragmentis an antibody fragment essentially equivalent to that obtained fromimmunoglobulins (typically IgG) by digestion with an enzyme pepsin at pH4.0-4.5. The fragment may be recombinantly produced. A “Fab” fragment isan antibody fragment essentially equivalent to that obtained byreduction of the disulfide bridge or bridges joining the two heavy chainpieces in the F(ab′)₂ fragment. The Fab′ fragment may be recombinantlyproduced. A “Fab” fragment is an antibody fragment essentiallyequivalent to that obtained by digestion of immunoglobulins (typicallyIgG) with the enzyme papain. The Fab fragment may be recombinantlyproduced. The heavy chain segment of the Fab fragment is the Fd piece.

Suitable polypeptide capture agents also include virtually any peptide,polypeptide, or protein that is capable of binding to an analyte ofinterest, or a small molecule such as a small organic molecule. In oneembodiment, the capture agent is an antibody that is capable of bindingto the analyte of interest. Suitable polypeptide capture agents may beobtained, for example, commercially, using recombinant methods, usingsynthetic production methods, or by purification from a natural source.Polypeptides include, for example, cell surface proteins, cell surfaceand soluble receptor proteins (such as lymphocyte cell surfacereceptors, steroid receptors), nuclear proteins, signal transductionmolecules, transcription factors, allosteric enzyme inhibitors, clottingfactors, enzymes (e.g., proteases and thymidylate synthetase,serine/threonine kinases, threonine kinases, phosphatases, bacterialenzymes, fungal enzymes and viral enzymes), proteins associated with DNAand/or RNA synthesis or degradation and the like. As described in moredetail below, where more than one capture agent is used, the captureagents can be, for example, isoforms of each other.

In a particular embodiment, where the analyte is a virus, the captureagent can be a cell surface receptor for the virus. For example, wherethe virus is HIV, the capture agent can be Dendritic cell-specificICAM-3 grabbing nonintegrin (DC-SIGN) or CD4. In another embodiment thecapture agent can be antibodies that are capable of binding viralantigens. For example, the antigen can be gp41 or gp120. The captureagent can be antibodies capable of binding host-derived antigens. Forexample, the antigen can be CD44, CD54, human leukocyte antigen (HLA)such as HLA-DR, or HLA-DRDPDQ.

The capture agent can also be a nucleic acid such as RNA or DNA, orpeptide nucleic acid. In one embodiment, the nucleic acid or peptidenucleic acid is capable of hybridizing to nucleic acid or peptidenucleic acid analyte. In addition, the capture agent can be an aptamer,a nucleic acid capable of binding to non-nucleotide analyte (e.g.,proteins, small organic molecules, or inorganic molecules). As usedherein, an aptamer can be either an RNA or a DNA chain composed ofnaturally occurring or modified nucleotides.

Suitable capture agents also include members of binding pairs. Suitablebinding pairs include, for example, biotin and avidin or biotin andderivatives of avidin (e.g., streptavidin and NEUTRAVIDIN™).

Capture agents can be bound to the surface or to the bead as describedbelow or by using standard techniques for attaching polypeptides,nucleic acids, protein A, and the like to surfaces.

Analytes

As used herein, the term “analyte” refers to, for example, the molecularstructure that is recognized by a capture agent. For example, the termanalyte can refer to the epitope recognized by an antibody, or caninclude that part of a ligand that is bound by a receptor. The termanalyte also includes larger molecules that contain a molecularstructure that is recognized by a capture agent. The analyte can be partof a cell, for example a cell surface protein. The analyte can be ananalyte of interest, chosen by the user (e.g., preselected). The analytecan be selected based on the ability to bind a capture agent ofinterest, for example in small molecule library screening.

In some embodiments, the analyte is a biological marker that may bepresent in a bioprocess fluid. As described herein, analysis ofbioprocess fluids can be performed using the apparatus described hereinin conjunction with a variety of commonly available commercial andspecially raised capture agents, depending on the biological marker ofinterest. Based on the information provided in Example IX, one ofordinary skill in the art would be able to select an appropriate captureagent for the biological marker of interest, and be able to analyze thebioprocess fluid for the presence and/or amount of the biologicalmarker. For example, capture agents that bind endotoxin and captureagents that bind protein A are commercially available.

The presence of one or more biological markers of a panel of biologicalmarkers can be assessed or detected. The panel of analytes can includeone or more analytes that are detected using a competition format asdescribed herein. The panel of analytes can include one or more analytesthat are detected using the sandwich assay format as described herein.In one embodiment, each analyte is detected using a separate cartridgeas described below In order to test a panel of one or more analytes, asingle sample can be divided into two or more aliquots. Each aliquot canbe tested for a different analyte, for example, using a differentcartridge for each analyte to be tested. In this manner, panels ofdifferent analytes may be tested without requiring that multiple samplesbe acquired and/or that different types of apparatus be employed to testthe detect the different analytes.

As used herein, the biological marker can be any component, other thanthe product molecule, present or suspected of being present in thestarting material or introduced into the bioprocess fluid duringprocessing. The nature of the biological marker or markers will be basedin part on the source of the bioprocess fluid, that is conditioned cellculture media, blood, milk, etc. In some embodiments, the biologicalmarker comprises host cell protein (HCP). HCP can be, for example, amixture of proteins or antigens that are produced and/or shed by thehost cell into the culture medium. In other embodiments, the biologicalmarker can be, for example, nucleic acids, proteinases, or viruses. Inother embodiments, the biological marker or markers can comprise one ormore milk antigens, serum antigens, or plasma antigens from the hostanimal. In addition, the biological marker can be an aggregate of theproduct of interest, such as aggregates of immunoglobulin, variants ofthe product molecule, deglycosylated versions of the product molecule,and the like.

In some embodiments, the one more biological markers comprises one ormore contaminants. Such contaminants can be introduced, oftenunintentionally, during the purification process. For example, protein Aor protein G columns are often used to purify antibody from a bioprocessfluid. However, a small amount of protein A or protein G may be shedfrom the column, thereby contaminating the bioprocess fluid. In someembodiments, the contaminants are microbes or microbial antigens, asdescribed, supra, including for example, protein A, protein, G,endotoxin, lipopolysaccharide, and other microbial toxins. In someembodiments, the one or more biological markers comprises one or morereagents that is added to the bioprocess fluid to facilitate isolationor purification of the product molecule, but that is removed in thefinal product.

As used herein, the one or more biological markers can be anycombination of biological markers described supra. Product molecules canbe distinguished from biological markers by a number of criteriaincluding, for example, antigenicity, molecular weight, pI,hydrophobicity, stability, and the like.

In one embodiment, the analyte of interest is a small molecule. Smallmolecules include organic or inorganic molecules having a molecularweigh of about 1000 g/mol or less. Typically, small molecule analytewill contain a single or only a few binding sites. Because a smallmolecule has a few or only one binding site, the present invention usescompetitive binding to detect and/or quantify small molecule analytes.

The small molecule can include, for example, steroids, lipids,carbohydrates, peptides, and heterocyclic compounds (e.g. bases,including co-factors such as FAD and NADH). The analyte (e.g., smallmolecule) can be part of a library of small organic molecules whichcomprise aldehydes, ketones, oximes, hydrazones, semicarbazones,carbazides, primary amines, secondary amines, tertiary amines,N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,thioethers, thioesters, disulfides, carboxylic acids, esters, amides,ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals,aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds and/or acid chlorides, preferably aldehydes,ketones, primary amines, secondary amines, alcohols, thioesters,disulfides, carboxylic acids, acetals, anilines, diols, amino alcoholsand/or epoxides, most preferably aldehydes, ketones, primary amines,secondary amines and/or disulfides and combinations thereof.

The analyte of interest can also be a polypeptide, a nucleic acid, acarbohydrate, a nucleoprotein, a glycopeptide or a glycolipid. Usefulanalytes include, for example, enzymes, steroids, hormones,transcription factors, growth factors, immunoglobulins, steroidreceptors, nuclear proteins, signal transduction components, allostericenzyme regulators, and the like. Analytes of interest can be obtained,for example, commercially, recombinantly, synthetically, or bypurification from a natural source. In preferred embodiments, theanalyte of interest is associated with a specific human disease orcondition.

Suitable growth factors include, for cytokines such aserythropoietin/EPO, granulocyte colony stimulating receptor, granulocytemacrophage colony stimulating receptor, thrombopoietin (TPO), IL-2,IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12, growth hormone, prolactin,human placental lactogen (LPL), CNTF, and octostatin. Suitable steroidsinclude, but are not limited to, estradiol, progesterone, testosterone,and derivatives thereof. Other suitable analytes include, for example,insulin, insulin-like growth factor 1 (IGF-1), epidermal growth factor(EGF), vascular endothelial growth factor (VEGF), placental growthfactor (PLGF), TGF-α and TGF-β), other hormones and receptors such asbone morphogenic factors, follicle stimulating hormone (FSH), andleutinizing hormone (LH), tissue necrosis factor (TNF), apoptosisfactor-1 and -2 (AP-1 and AP-2), and mdm2.

In one embodiment, the analyte is a cardiac marker. Cardiac markers arewell known in the art and include, for example, c-troponins I and T,myoglobin, creatin kinase MB (CK-MB), and ischemia modified albumin. Inone embodiment, the present invention can be used to detect a panel ofanalytes in order to assess a patient's condition. In one embodiment, apanel of cardiac markers is tested. The panel can include, for example,c-troponin-I, CK-MB, and myoglobin analytes.

In another embodiment, the analyte is a marker for vial infection.Markers for viral infection include, for example, inflammatory markers,circulating viral proteins, CD4+ cells, liver enzymes, and anti-virusantibodies.

The analyte of interest can also be a therapeutic drug where it would beuseful to measure the levels of the drug in a patient sample, forexample for drug management purposes or patient compliance. Suitabletherapeutic drugs include, but are not limited to protease inhibitorsand immunosupressants. Suitable protease inhibitors include ageneraser,reyataz, lexiva, telzir, crixivan, kaletra, viracep, norvi, invirase,aortovase, aptivus and the like). Suitable immunosuppressants includecyclosporin, tacrolimus (FK-506), rapamycin, mycophenolic mofetil andthe like.

The analyte of interest can be a pathogen or microbe, such as bacteriaor bacterial spores, viruses, parasites, prions or other pathogens ortheir cell wall or surface components such as gram-positivepeptidoglycans, lipoteichoic and teichoicacids, and gram-negativeendotoxin (e.g.) lipopolysaccharide). Bacterial analytes include, forexample, Shigella sp. such as Shigella dysenteriae, Campylobacter sp.such as Campylobacter jejuni, Enterococcus sp. such as Enterococcusfaecalis, Bacillus anthracis, Yersinia pestis, Bordetella pertussis,Streptococcal species, Staphylococcus aureus, Mycobacteriumtuberculosis, Clostridium difficile, Clostridium tetani, Clostridiumbotulinum, Escherichia coli, Salmonella thyphimurim, Salmonellaenterica, Chlamydia species, Treponema pallidum, Neisseria gonorrhoeae,Borrelia burgdorferi, Vibrio cholerae, Corynebacterium diphtheriae, andHelicobacter pylori. Parasites include, for example, Giardia, malariaand crytosporidia. Viral analytes include, for example, Rhinovirus,Yellow Fever, Group B Coxsachieviruses, (CB1, CB2, CB3, CB4, CB5, andCB6), Canine Parvovirus (CPV), Herpes Simplex virus type 1 (HSV1),Vaccina Virus, T4-like virus, Adenovirus, Influenza B virus, InfluenzaA, Avian flu, rhinovirus, coronavirus (e.g., SARS), HumanImmunodeficiency virus (HIV), Hepatitis viruses, Herpes virus, West NileVirus, and Ebola virus.

As described above, in some embodiments, more than one analyte isdetected. In one embodiment, the analytes have the same molecularstructure. The analytes can be, for example the same molecular speciesof interest. In another embodiment, the different analytes (alsoreferred to herein as the first and second analytes) are part of alarger molecule. Therefore, the analyte can be a particular binding site(such as an epitope) attached to or contained within a larger molecule.As such, the different analytes being detected can be part of differentmolecules.

Analytes can be bound to the surface or to the bead as described belowor by using standard techniques for attaching polypeptides, nucleicacids, and the like to surfaces. In one embodiment, the analyte isindirectly bound to the surface. The analyte can be indirectly bound tothe surface, for example, by coating the surface with a first member ofa binding pair. The analyte is bound or attached to a second member ofthe binding pair and then the analyte is bound to the surface via theinteraction between the first and second members of the binding pair.Suitable binding pairs include, for example, biotin and avidin or biotinand derivatives of avidin such as streptavidin and NEUTRAVIDIN™.

Assay Formats

As described above, in one embodiment of the present invention, aplurality of magnetic particles is introduced into a fluid chamber. Themagnetic particles are coated with a first capture agent capable ofbinding the analyte and at least one surface of the fluid chambercomprises an acoustic device that has been coated with a second captureagent capable of binding to the competitor molecule. The surface can becoated with the second member of the binding pair using any suitablemethod. For example, the surface can be coated with biotin as describedbelow and then the biotinylated surface can be exposed to avidin or aderivative of avidin, and the second capture agent can be linked tobiotin.

In another embodiment, a plurality of magnetic particles and acompetitor molecule are introduced into a fluid chamber. The magneticparticles are coated with a first capture agent capable of binding theanalyte. At least one surface of the fluid chamber comprises an acousticdevice that has been coated with a second capture agent capable ofbinding to the competitor molecule. In one embodiment, the competitormolecule comprises the analyte linked or bound to a tag, and the secondcapture agent is capable of binding to the tag. The tag can be anymoiety that can be recognized by a capture agent. In one embodiment, thetag is one member of a binding pair and the capture agent is the othermember of the binding pair. For example, the tag can be biotin, and thecapture agent can be avidin, streptavidin, or NEUTRAVIDIN™. In thisembodiment, the biotin is linked to the analyte using known methods forlinking molecules to biotin, to form the competitor molecule. Thesurface can be coated with the second member of the binding pair usingany suitable method. For example, the surface can be coated with biotinas described below and then the biotinylated surface can be exposed toavidin or a derivative of avidin.

In another embodiment, the competitor molecule comprises two or moreanalyte molecules bound to a carrier, and the second capture agent iscapable of binding the analyte. The carrier can be any molecule to whichtwo or more analyte molecules can be linked or bound. The carrier can bea protein, a nucleic acid, or other polymer. In one embodiment, thecarrier is an albumin, such as bovine serum albumin. In anotherembodiment, the carrier is horseradish peroxidase. In this embodiment,two or more analyte molecules can be linked to the carrier as describedbelow, or known linking technology can be used to form the competitormolecule. The surface can be coated with the capture agent as describedbelow.

According to one embodiment of the method of the present invention, theplurality of particles can be exposed to the sample using a variety ofdifferent orders of exposure. For example, in one embodiment, theplurality of particles is exposed to the sample and then introduced intothe fluid chamber. The sample may be concentrated prior to introducingthe sample-exposed particles into the fluid chamber. The sample may beconcentrated by, for example, removing the particles from the solutionand resuspending the particles in a smaller volume of liquid.

In another embodiment, the sample is introduced into the fluid chamberprior to introducing the plurality of particles. In still anotherembodiment, the plurality of particles is introduced into the fluidchamber prior to adding the sample to the fluid chamber.

In another embodiment, as described above, the plurality of particles isexposed to the sample and to a competitor molecule. The plurality ofparticles can be exposed to the sample and to the competitor molecule ina number of different orders. For example, in one embodiment, theplurality of particles is exposed to the sample and to the competitormolecule and then introduced into the fluid chamber. In anotherembodiment, the sample and the competitor molecule are introduced intothe fluid chamber prior to introducing the plurality of particles. Instill another embodiment, the plurality of particles is introduced intothe fluid chamber prior to adding the sample and/or the competitormolecule to the fluid chamber.

Control Signal/Normalization

In another embodiment, the signal output of the acoustic device inresponse to sample exposure is compared to or normalized with a controlsignal. The control signal can be provided to or obtained by the user.For example, the control signal can be a value provided to the userbased on the specific analyte, capture agent, or particular model,version or type of device being used. In one embodiment, the controlsignal is obtained on a lot basis. For example, the control signal canbe a signal that is representative of a particular lot of analyteacquired by the user. The representative signal can be, for example,experimentally derived before, during or after testing of a sample. Insome embodiments, the control signal is a standard curve that isobtained by, for example, analyzing known quantities of analyte with aspecific capture agent and specific version of an acoustic device.

In another embodiment, the control signal is obtained on a use basis.For example, a unique control signal can be obtained each time aparticular analyte and/or capture agent is tested on a particularacoustic device. In one embodiment, the control signal is obtained usingthe same analyte and/or capture agent, however, in the absence ofsample. The control signal can be obtained by introducing a secondplurality of particles into the fluid chamber that are coated with acapture agent. At least one surface of the fluid chamber comprises anacoustic device that has a second analyte bound thereto. Signal outputby said acoustic device is monitored to determine a control signal to beused in subsequent testing.

As described below for multiplexing, the different pluralities ofmagnetic particles can be exposed to the same or different surfaces ofthe fluid chamber.

Acoustic Devices

Acoustic devices couple to fluids predominantly through acousticinteraction between the device and the fluid. Typical acoustic devicesinclude surface acoustic wave devices, flexural plate wave devices, lambwave devices and cantilever devices. Acoustic devices also couple tofluids through some viscous interaction between the device and thefluid, however, the coupling is predominantly acoustic coupling. Viscousinteraction devices couple to fluids predominantly through viscousinteraction between the devices and the fluid. Typical viscousinteraction devices include quartz microbalance (QCM) devices, shearharmonic surface acoustic wave devices, and acoustic plate mode devices.The term “surface acoustic wave” refers to the manner in which energy iscarried in the device structure rather than how the device couples tothe fluid. Acoustic devices are devices where fluid interacts over asubstantial area of a plane of the device. Acoustic devices respond withsubstantial out of plane motion that couples acoustically to fluid inproximity to the plane of the device (i.e., kinetic energy, potentialenergy and losses are carried predominantly in the fluid). Viscousinteraction devices respond primarily with in-plane motion that does notcouple acoustically to fluid in proximity to a plane of the device.

For applications involving, for example, the detection andquantification of biological or chemical substances in a fluid, thecoupling between an acoustic device and a fluid is typically betweenabout 100 nm and about 10 microns in thickness relative to the plane ofthe device where the coupling between a viscous interaction device and afluid is between about 10 nm and about 100 nm in thickness relative tothe plane of the device.

Surface acoustic wave devices and shear harmonic surface acoustic wavedevices both carry energy in their respective structures in similarmanners. Surface acoustic wave devices acoustically couple significantlyto fluids while shear harmonic surface acoustic wave devices couple tofluids predominantly through viscous interaction.

One embodiment of an analyte detection system 100, constructed accordingto the invention, is shown in FIG. 1A. The system 100 includes a networkof channels 102 for transporting various test solutions (also referredto herein as “test fluids” or “fluids”) through an FPW device 104. Thefollowing U.S. patents and patent applications, all of which are herebyincorporated by reference, describe examples of the various types of FPWdevices suitable for use in the present invention: U.S. Pat. No.5,129,262, U.S. Pat. No. 5,189,914, U.S. Pat. No. 6,688,158 B2, U.S.patent application Ser. No. 10/324,685, U.S. Pat. No. 5,668,303, U.S.Pat. No. 5,836,203, and U.S. Patent Application 20040038195.

For example, U.S. Pat. No. 5,129,262 describes an ultrasonic sensor thathas a thin planar sheet of material forming a Lamb wave propagationmedium. Lamb waves, also known as plate-mode waves, can propagate onlythrough a material of finite thickness. In contrast to surface acousticwaves (SAWs), which require a propagation medium having a thickness onthe order of hundreds of times the wavelength of the propagating SAW,Lamb waves require a propagation medium which is at most only severalwavelengths thick, and typically only a fraction of the wavelength ofthe propagating Lamb wave. The thickness of the sheet is no greater thanabout twenty microns. A Lamb wave generator generates Lamb waves in theplanar sheet, and an output device produces an electrical signal thatrepresents the propagation characteristics of the Lamb waves propagatingalong the sheet. A measuring device measures selected characteristics ofthe output electrical signal. The planar sheet has some physicalcharacteristics that depend upon the value of a measurand acting on thesheet, and those physical characteristics consequently determine thepropagation characteristics of the Lamb waves that propagate along thesheet. Since the electrical signal from the output device represents thepropagation characteristics, the electrical signal also represents thevalue of the measurand acting on the sheet.

The Lamb wave device described in U.S. Pat. No. 5,129,262 can beemployed, for example, in biological sensing. The planar sheet describedabove can be pre-coated with antibody molecules, so that the frequencyof the device changes upon immersion in or contact with a liquid thatcontains the corresponding antigen. Antigen-antibody attachment at thesurface of the propagation medium acts to alter the wave velocity of theLamb waves in the sheet. The change in wave velocity causes theoscillation frequency to change in a delay line oscillator form of thedevice. Also, the sheet may be made of a porous and permeable material,allowing the coating of antibody molecules over a greater surface areaof the sheet and also allowing the antigen-containing liquid to beflowed through the membrane, in order to speed up the antigen-antibodyattachment. Other biological interactions may also be sensed, andadditional applications include immunoassay, clinical laboratorytesting, in vivo biomedical monitoring, and biomedical research.

The test solutions used in the described embodiment, for example ablocking solution 106, a sample 108, and a buffer 110, are sourced fromreservoir containers 112. The channel path from each of the reservoirs112 is gated with a valve 114 to control the flow of a particular testsolution to a combination point 116 leading to an entry port 118 of theFPW device 104. The test solution flows through the FPW device 104 andexits via an exit port 120, which leads to a pump 122. The pump 122draws the test solution through the network of channels 102 and throughthe FPW device 104, and directs the test solution to a waste receptacle124.

FIG. 1B shows another embodiment of the analyte detection system 100.This embodiment packages the FPW device 104 and its associated fluidchamber 160 as a cartridge 103, i.e., a consumable component that can beremoved and replaced. Some embodiments may include a fluid controldevice 101 such as a plug, obstruction or baffle that alters the flowthrough the device 104. In one embodiment, the fluid control device 101operates to cause the fluid flow through the device 104 to pass closerto the sensor surface 143 than if the fluid control device 101 was notpresent. Further, the source of input test solutions is shown as aninput fluid chamber 105 that has an outlet 107 for directing the testsolutions into the inlet 109 of the cartridge 103. In some embodiments,the magnetic particles are initially located in the input fluid chamber105 and the fluid containing analyte is mixed with the magneticparticles in the input fluid chamber 105 and then directed into thecartridge 103 in which the FPW device 104 is located. The magneticparticles may be combined within the input fluid chamber 105 with thefluid containing analyte by a device (e.g., by the action of a pump or amagnetic agitator). FIG. 1B further shows an output fluid chamber 111with an inlet 113 that receives fluid from the outlet 115 of thecartridge 103. This output fluid chamber 111 may include one or more ofthe fluid control devices described herein, and it may include one ormore mechanisms for storing and/or treating waste fluid.

In at least one embodiment, the junction where the outlet 107 of theinput fluid chamber 105 meets the inlet 109 of the cartridge 103 isconstructed and arranged to allow repeatable connection anddisconnection. Similarly, the junction where the outlet 115 of thecartridge 103 meets the inlet 113 of the output fluid chamber 111 isconstructed and arranged to allow repeatable connection anddisconnection. In some embodiments, these junctions are constructed andarranged to require tools for connection and disconnection, such asthreaded couplings that require a wrench or other such tool to affectthe coupling and decoupling. In other embodiments, these junctions areconstructed and arranged to allow quick and easy manual connection anddisconnection, without any extra tools or accessories. Such couplings,both requiring and not requiring tools, are known in the art. In someembodiments, there are multiple input fluid chambers and output fluidchambers. In some embodiments, one or more input and/or output fluidchambers are part of the cartridge 103. Further, in some embodiments,one or more sources of magnetic flux are part of the cartridge.

The FPW device 104 is shown in more detail in FIG. 2 and FIG. 2A. In anFPW device 104, strain energy is carried in bending and tension in thedevice. In some embodiments, it is desirable for thethickness-to-wavelength ratio of the FPW device 104 to be less than one,and in some cases much less than one. In general, the wavelength “λ” ofthe FPW device 104 is approximately equal to the pitch of theinterdigitated electrodes as described herein. In one embodiment, thethickness-to-wavelength ratio of the FPW device 104 is 2 μm/38 μm. Inother embodiments, the FPW device 104 is designed to isolate aparticular mode (e.g., any mode from the zero^(th) order mode to higherorder modes) or bandwidth of modes associated with the device. Forexample, an FPW device 104 having a thickness/wavelength of 2 μm/38 μmas described above would isolate the 80^(th) mode of the FPW device 104.The FPW device 104 can be designed to achieve this effect by selecting aparticular pattern for the interdigitated electrodes deposited on thedevice. In one embodiment, the FPW device 104 is rectangular in shape.The FPW device 104 can, alternatively, be circular or elliptical, orsome other planar shape.

In general, the FPW device 104 is constructed from a silicon wafer 130,using micro-fabrication techniques known in the art. In the describedembodiment, a cavity 132 is etched into the wafer 130 to produce a thin,suspended membrane 134 that is approximately 1.6 mm long, 0.3 mm wideand 2 μm thick. The overall wafer 130 thickness is approximately 500 μm,so the depth of the cavity 132 is just slightly less than the wafer 130thickness. A 0.5 μm layer 136 of aluminum nitride (AlN) is deposited onthe outer surface (i.e., the surface opposite the cavity 132) of themembrane 134, as shown in the expanded view insert of FIG. 2A. Two setsof inter-digitated metal electrodes 138 are deposited upon the AlNlayer. A thin layer 140 of gold (approximately 500 angstroms) isdeposited on the inner surface (i.e., the surface facing the cavity 132)of the membrane 134 to facilitate immobilization of capture agents(described in more detail below). In some embodiments, the first of thetwo sets of the interdigitated electrodes is an actuating or launchingportion that is located on the left half side (as viewed in FIG. 2) ofthe membrane 134. The second of the two sets of interdigitatedelectrodes is a sensing or receiving portion that is located on theright half side (as viewed in FIG. 2) of the membrane 134. In someembodiments, the two sets of the interdigitated electrodes are locatedside-by-side along the length (viewed from left to right in FIG. 2) ofthe membrane 134, one of the two sets is the actuating or launchingportion and the second of the two sets is the sensing or receivingportion.

In operation, instrument/control electronics 126 (referring to FIG. 1A)apply a time-varying electrical signal to at least one set of electrodes138 to generate vibrations in the suspended membrane 134. Theinstrument/control electronics 126 also monitor the vibrationalcharacteristics of the membrane 134 by receiving a sensor signal from atleast a second set of electrodes 138. When liquid is in contact with thecavity side 132 of the membrane 134, the maximal response of the platestructure is around 15-25 MHz. The instrument/control electronics 126compare a reference signal to the sensor signal from the second set ofelectrodes to determine the changes in the relative magnitude and phaseangle of the sensor signal as a function of frequency. Theinstrument/control electronics 126 interpret these changes to detect thepresence of the targeted analyte. In some embodiments, theinstrument/control electronics also determines, for example, theconcentration of the targeted analyte on the inner surface of themembrane 134.

Capture agents targeting the analyte of interest are immobilized on thethin layer of gold 140 covering the inner surface of the membrane 134,as described above. The surface can be coated with a suitable linkingcompound. Suitable linking compounds are commercially available. In oneembodiment, the linking compound comprises biotin PEG disulfide, asdescribed below. In another embodiment, thiol-terminated alkyl chainsare linked to the gold surface forming a self-assembled monolayer (SAM).A fraction of the SAM chains are terminated with reactive groups (e.g.,carboxyl) to allow covalent linking of capture agents to the SAM chainsusing biochemical process steps known in the art. The remainder of theSAM chains are terminated with non-reactive groups, preferably ones thathave a hydrophilic character to resist nonspecific binding (e.g.,oligomers of ethylene glycol). Other surface chemistries are describedin the literature and can be used to produce a capture surface.

The FPW device 104 is packaged to allow electrical connections to theelectrodes 138 on the outer surface of the membrane 134. Additionally,the FPW device 104 is mechanically supported by a channel block 142, toallow for the inner surface of the membrane 134 to contact the testsolutions and an interface is provided for contacting the sensor surface143 with the liquid sample. The channel block 142 creates a path (fluidchamber 160) for the test solutions to flow from an input port 118, pastthe inner surface of the membrane 134 and then out of an exit port 120.A seal 144 is formed between the FPW device 104 and the channel block142 to prevent test solutions from escaping from the channels 102 formedwithin the combination of the FPW device 104 and the channel block 142.The channel block 142 thus forms a fluid chamber, of which the FPWdevice 104 comprises one of the interior walls.

The channels 102 through the combination of the FPW device 104 and thechannel block 142 are approximately 0.5 mm in diameter. The channelblock 142 can be formed from a variety of materials, including plastic,metal or ceramic, among other materials.

The system 100 includes one or more fluid control devices for changingat least one fluid property, such as flow, pressure, or trajectory toname a few, within the system 100. The pump 122 and valves 114 shown inFIG. 1A that direct and control the flows of various test solutionsthrough the device and over the sensor surface 143 (as required toexecute a test protocol) are all examples of fluid control devices. Ingeneral, a fluid control device changes the at least one fluid propertyin the vicinity of at least one surface within the fluid chamber 160 ofthe device 104. Generally, this is done to distribute the magneticparticles along at least a portion of the sensor surface 143. Asdescribed above, in some embodiments the fluid control device is a pump(e.g., a peristaltic pump, centrifugal pump, rotary pump,electro-osmotic pump). In some embodiments, the pump is located on theentrance side of the fluid chamber, and in other embodiments the pump islocated on the exit side of the fluid chamber. In some embodiments, thedevice is a flow diverter (e.g., a plug, obstruction wall or baffle)that is disposed relative to the fluid chamber to alter the fluid flowin the vicinity of the at least one interior surface of the fluidchamber.

Referring to FIG. 1A, a single pump 122 is positioned on the waste sideof the FPW device 104. Suction that the pump 122 generates draws buffer110 or analyte in the sample 108 from their respective reservoircontainers 112 on the supply side of the FPW device 104. Valves 114 arepositioned on the supply side of the device 104 to control which testsolution is directed over the sensor surface 143 at any time during thetest protocol. The pump 122 controls the flow rate of the test.

A device for regulating temperature (e.g., a thermoelectric cooler) maybe associated with the FPW device 104 and channel block 142. Thisreduces the impact of variable environmental conditions on the FPWdevice 104 output by maintaining the device 104 at a relativelyconstant, known temperature. In an alternative embodiment, a temperaturesensor is included within the system 100, for example as part of the FPWdevice 104. The sensor signal from the FPW device 104 is scaled, at aspecific instant in time (or during a period of time), based on theoutput of the temperature sensor, in order to produce a signal that isindependent of the effects of temperature variations. This scaling couldbe done based on a mathematical model, or an analytical model, or somehybrid combination of a mathematical and analytical model.

In some embodiments of the system 100, a filter is included in the pathof the test solution to selectively filter particles (e.g., magneticparticles and biological materials) of a particular size to prevent themfrom entering the fluid chamber. By way of example, a particular testingprotocol may include steps for changing the filter during the test. Thiswould allow different types (i.e., sizes) of analytes and magneticparticles to be directed into the fluid chamber, and thereby tested bythe system 100, during different portions of the test.

In one embodiment, magnetic particles (e.g., paramagnetic orsuper-paramagnetic beads or microspheres), which have their surfacescoated with a capture agent, are mixed with a sample containing theanalyte. After a prescribed mixing time analyte-particle complexes 146result as do particles 147 that have bound nonspecific materials andparticles 148 that have bound nothing. The particles 146, 147 and 148are located in the sample reservoir 112.

The system 100 further includes a magnetic field inducing structure 150for producing magnetic flux in the vicinity of the membrane 134. In FIG.1A, the source of magnetic flux is a retractable magnet 150 arranged tonormally be in close proximity to the membrane 134 of the FPW device104. When the magnet 150 is in close proximity to the membrane 134, themagnet 150 produces a significant gradient magnetic field in thevicinity of the membrane 134. Under control of the instrument/controlelectronics 126, the retractable magnet 150 can be retracted away fromthe membrane 134 by a distance sufficient to substantially reducemagnetic fields in the vicinity of the membrane 134. In one embodiment,when in close proximity to the membrane 134, the magnet 150 is situatedapproximately 200 μm from the sensor surface 143 of the membrane 134. Inanother embodiment, when in close proximity to the membrane, the magnet150 is situated between about 50 μm to about 100 μm from the sensorsurface 143 of the membrane 134.

When the magnet 150 is in close proximity to the membrane 134, themagnet 150 provides a source of magnetic flux to draw the magneticparticles from the sample to the sensor surface 143. Theanalyte-particle complexes 146, as well as particles 147 withnonspecifically bound material and particles 148 with nothing bound,migrate from the liquid sample until they encounter the sensor surface143. The analyte binds with the capture agent on the sensor surface 143.Thus, the analyte forms a link between the magnetic particle and sensorsurface. The particles 147 with non-specifically bound material andparticles 148 with nothing bound are held at the sensor surface 143 bythe magnetic field. Additionally, weak binding forces can act betweenthe particles 146, 147, and 148 and the sensor surface 143. During thewash step of the protocol (described in more detail below), the magnet150 is retracted to reduce the magnetic force experienced by theparticles that have accumulated at the sensor surface 143. The wash flowrate is increased to remove particles 147 and 148 that are not bound tothe surface by analyte. Since the particles 147 with nonspecificallybound material as well as particles 148 with nothing bound are moreweakly linked to the sensor surface 143 than the analyte-particlecomplexes 146, they release from the sensor surface 143 at a lower washflowrate (and corresponding hydrodynamic force). Hence, removing themagnet 150 (i.e., substantially reducing the magnetic force experiencedby the particles 146, 147, and 148 at the sensor surface 143) is used todistinguish between particles with analyte 146 from those without(particles 147 and 148). One technique for engaging and retracting themagnet 150 is to mount it on a carriage (not shown) that is actuated bya cam system (not shown).

The magnet 150 material, geometry and distance from the sensor surface143 determine the field shape and field gradient, and therefore, theforce that the analyte-particle complexes 146 experience. High strengthpermanent magnets for use as the retractable magnet 150 are availablecommercially. For example, 1 mm diameter cylindrical NdFeB magnets canbe purchased from several vendors (e.g., Dexter Magnetic Technologies).In one embodiment, a 1 mm diameter and 5 mm long NdFeB magnet 150 ispositioned within 0.1 mm of the sensor surface 143 when engaged. Whenretracted the magnet 150 is at least 0.5 mm from the sensor surface 143.Since the membrane 134 of the FPW device 104 is very thin (2 μm) andmade of nonmagnetic materials (e.g., silicon, aluminum nitride or gold),the membrane 134 does not significantly perturb the magnetic field onthe sensor surface 143 side of the device 104. As a result, very highmagnitude magnetic fields and large field gradients can be achieved, asis necessary for high collection efficiencies.

The sample flow rate through the channels 102 is determined (e.g.,specified by an operator) by the residence time necessary for goodcollection efficiency. The sample flow rate is adjusted so that theaverage velocity over the sensor surface 143 is between about 1 andabout 5 mm/s. With an iron oxide paramagnetic particle with a diameterof approximately 3 μm, collection efficiencies approaching 50% can beachieved.

Other configurations of the source 150 of magnetic flux (i.e., themagnet) may be used. For example, an electromagnet can be used insteadof a permanent magnet. The electromagnet includes pole pieces thatextend to focus the field flux near the sensor surface 143 of the device104.

Alternatively, a magnetizable material can be fashioned and positionedadjacent to the sensor surface 143 (within 0.1 mm), and a separatemagnet combined with an open face of the magnetizable material to inducea magnetic field in the magnetizable material. The magnetic fieldinduced in the material serves to locate desirable field gradients nearthe sensor surfaces 143. In this way, large, low cost magnets can beused, and a single magnet can be used to address multiple sensors,depending on the fashioning of the material. Examples of usefulmaterials for this purpose are pure iron, high mu metals such as alloy49 (high nickel content iron), sna silicon steels (1-2% silicontypical). An advantage of using such a magnetizable material with anassociated magnet is that the sensor assembly can be simplified,allowing lower cost manufacturing. A low precision actuator can be usedfor engaging and retracting the magnet since the magnet need onlycontact the ferromagnetic core or be fully withdrawn. In the describedembodiment where the magnet 150 is positioned in close proximity to thesensor surface 143, a higher level of precision is required to achievegood assay repeatability. Although there is some loss of field strengthwith this approach, it is still possible to design the overall system toachieve good capture efficiencies (e.g., >10%).

The shape of the tip of the field inducing structure (e.g., magnet orferromagnetic material) may be tailored to enhance and/or concentratethe field gradient at the surface. Since the size of the FPW device 104(e.g., 0.3 mm×1.6 mm) is typically smaller than conventionally formedmagnets or machined inductors, the portion of the field inducingstructure adjacent to the membrane 134 can be tapered to concentrate themagnetic field in one or more locations on the sensor surface 143.Tapering the tip acts to increase both the local field magnitude and thelocal field gradients. For example, a wedge-shaped tip is well suited tothe current FPW device geometry.

One embodiment of the system 100 includes an optional second source 150a of magnetic flux that opposes or partially opposes the first source150 of magnetic flux. This second source 150 a of magnetic flux can beused to dislodge some of the magnetic particles that have adhered to thesensor surface 143. It may, for example, dislodge magnetic particles 148that do not have any bound analyte; they would not be as stronglyattached to the sensor surface 143 as the particles 146 that do havebound analyte. In some embodiments, the first source 150 of magneticflux is turned off or moved away from the sensor surface 143 and then,the second source 150 a of magnetic flux is positioned relative to theat least one surface of the fluid chamber to selectively remove magneticparticles. This may be done, for example, to remove magnetic particles148 that do not have any bound analyte and therefore they are not asstrongly bound to the sensor surface 143. This would achieve a similareffect as increasing the flow of fluid to remove magnetic particles 148that do not have any bound analyte.

Controlling the distribution of the analyte-particle complexes 146 onthe surface 143 of the device 104 can improve the device performance,since the device 104 has a suspended membrane 134 and not all parts ofthe membrane 134 contribute equally to the moving mass of the detectableresonance. For example, the system 100 can be constructed and arrangedto distribute the analyte-particle complexes 146 within one third of theFPW device 104 width along the middle two-thirds of the centerline ofthe long axis of the membrane 134. Taking into account flow fieldeffects, the shape of the tip of the field-inducing structure (e.g.,magnet 150) can be such that the field magnitude and field gradientincrease in the direction of the flow over the sensor membrane 134. Thatis, analyte-particle complexes 146 in the downstream regions, where theboundary layer is partially depleted of analyte, experience a higherfield and field gradient than do analyte-particle complexes 146 in theupstream regions.

In general, the system 100 can be constructed and arranged toconcentrate magnetic particles in one or more particular regions of thesensor surface 143. The response of the device 104 may not be uniformover the sensor surface 143 due to characteristics of the fabricationmaterials or the specifics of the sensor design. Thus, high sensitivityregions of the device 104 may be non-uniform and asymmetrical withrespect to the long and short axis centerlines of the device 104. Thus,the tip of the field inducing structure may be shaped to concentratemagnetic particles in the region or regions of highest sensitivity.

Varying the flow rate through the device 104 can also be used to achievea more uniform coverage of analyte-particle complexes 146 for a givenmagnetic field distribution. For a given field, magnetic particlesinteract with the sensor surface 143 as determined by the bulk fluidflow rate, much like a ballistic object might fall in the presence ofthe gravity body force. In this case, however, the magnetic inducedforce dominates. By varying the flow rate, the analyte-particlecomplexes 146 can be caused to interact with the sensor surface 143 atsubstantially different locations along the stream-wise flow direction.Furthermore, as the magnetic particles pile up (a non-desirableoccurrence if they are to be exposed to the sensor surface 143) the flowcan be reversed and subsequently pulsed forward in order to pull thepile over and thus communicate more particles with the sensor surface143. In one embodiment of the system 100, selective location of themagnetic particles along the sensor surface 143 is achieved byselectively altering, over the course of the detection protocol, eitherone or both of the magnetic flux source and the property or propertiesof the fluid flow along the sensor surface 143.

One embodiment of the system 100 includes a device (e.g., optical,magnetic) for characterizing at least one property of the magneticparticles that are attached or attracted to the sensor surface 143. Thisdevice could be an integral part of the FPW device 104, or it could be apart of the magnet 150, or it could be a discrete component apart fromother components of the system 100. Such a device may be used to detectthe presence of the particles, and also to determine parameters relatedto the particle, for example, the size, quantity, concentration, ordensity of the particles that are attracted to the sensor surface 143.

One embodiment of the system 100 includes an identification device forallowing an operator or computer to identify the system 100 or aparticular component of the system for tracking usage of the system orcomponent. The identification device may include a symbol or image suchas a bar code, an identification number, or other identifying mark. Theidentification device may include an actual component, passive oractive, such as an RFID tag, an integrated circuit or other suchcomponent known in the art for providing identifying information. Manysuch devices are known in the art, although any contemplatedidentification device may be used.

A general detection protocol 200 for using an FPW device 104 incombination with analyte-particle complexes 146 for detection ofbiological analytes is shown in FIG. 3.

The first step 202 of the detection protocol 200 is acquiring andpreparing 202 the analyte sample. Various preparation processes may needto be performed prior to testing, depending upon the particular type ofanalyte being tested. For example, to detect microbes in food (e.g., E.coli in ground beef), a sample would be first mixed with enrichmentbroth, stomached, incubated and filtered. For detecting proteins inblood, the sample would first be filtered or centrifuged and the serumseparated. Specific examples of test protocols that include samplepreparation steps are described herein.

The next step 204 of the detection process is mixing affinity-coatedparamagnetic particles (i.e., beads) with the prepared analyte sample.Paramagnetic or super-paramagnetic particles are available commerciallyfrom a number of vendors (e.g., Dynal Biotech, Oslo, Norway). Typicaldiameters range from 50 nm to 10 μm, and such particles can be procuredalready coated with affinity agents (e.g., antibodies, proteins, andnucleic acid probes) targeting a variety of analytes (e.g., cells,proteins and nucleic acids). Alternatively, the particles can bepurchased with various reactive chemical groups (e.g., epoxy, carboxyl,and amine) in order to attach a capture agent of choice. Standardbiochemical protocols are available for this purpose.

The sample with paramagnetic particles added is agitated 206 for anamount of time determined by the particular analyte and capture agent.During this process, the particles bind with analyte so that the analyteis captured on the particles. In some cases, the sample can be testeddirectly at this point. But, in other cases it is advantageous toperform separation steps 208 to isolate the analyte bound to theparticles from the rest of the original sample. These separation steps208 reduce interference from other biological material in the assay.Manual or automated equipment for performing such separation steps isavailable commercially (e.g., Dexter Magnetic Technologies, DynalBiotech). The basic process uses a magnet to localize the paramagneticparticles on a surface so that the majority of the sample liquid can beaspirated. The magnet (e.g., magnet 150 of FIG. 1A) is then removed, andclean buffer solution is added to re-suspend the particles.

In one embodiment, a baseline step 210 is executed prior to testing theprocessed analyte sample with the FPW device 104. During the baselinestep 210, a reference solution 106 is flowed through the system torinse/block the sensor 104, and the instrument/control electronics 126excite the device 104 and records the resulting initial baseline signalfrom the device 104.

A sample transport step 212 follows the baseline step 210. The sample108 containing the analyte-particle complexes 146 is flowed over thesensor surface 143 with the magnet 150 engaged. Analyte-particlecomplexes 146 are collected on the sensor surface 143. After aprescribed volume of sample 108 has flowed through the device 104, themagnet 150 is retracted to release the particles 147 and 148 from thesensor surface 143 that do not have bound analyte, and the flow isswitched to a wash solution (e.g., buffer solution 110). The flow rateof the wash solution is increased to help remove loosely bound particles147 and 148, as well as other material in the sample that may have boundto the sensor surface 143

An acquisition step 214 follows the sample transport step 212. Referencesolution 106 is again run through the device 104, and theinstrument/control electronics 126 excite the device 104 to acquire andrecord a final baseline signal from the device 104.

The system 100 determines the amount of analyte accumulated on thesensor surface 143 during the transport step 212 by comparing 216 theinitial baseline signal and the final baseline signal, which correspondto the vibrational characteristics of the FPW device 104 in thereference solution before and after, respectively, the acquisition step214. Analyte-particle complexes 146 bound to the sensor surface 143change the vibrational characteristics of the FPW device 104, and theamount of change to the vibrational characteristics correspond to theamount of analyte-particle complexes bound to the sensor surface 143.

FIG. 4 shows the change in the signal from multiple FPW devices 104 as afunction of time for an exemplary detection protocol. The square symbolscorrespond to a device 104 exposed to an analyte; the triangles andstars correspond to negative controls (in which there is no analyte onthe beads). The data shown in FIG. 4 (and FIG. 5 as described below)represent an exemplary detection protocol for which the analyte is E.coli bacteria, or generally a cellular analyte.

In this particular experiment, the frequency of a resonant peak wastracked. FIG. 4 shows that the resonant frequency of the FPW device 104decreases as the magnetic particles 146, 147 and 148 accumulate on thesurface. Once the magnet 150 is removed and some of the magneticparticles are washed away, the frequency of the device 104 increases.Eventually, the system establishes a final baseline that can be comparedto the initial baseline taken at the start of the test, or to that of acontrol.

FIG. 5 is a summary plot showing the final signal change detected as afunction of original analyte concentration.

Other detection protocols may be used with the system 100 describedabove. Individual steps can be eliminated or added depending on therequirements of a specific application or analyte. For example, FIG. 6shows a time evolution plot for a detection protocol, similar to the oneshown in FIG. 4. FIG. 6, however, depicts a detection protocol for a PSAassay, which demonstrates the capability of the system 100 for detectingproteins. Also, the flow direction can be reversed during the protocol.For example, reversing the flow direction may be useful for washingnonspecifically bound material from the device, or for making moreefficient use of the available sample.

Another variation in the detection protocol includes alternating betweenwash steps and binding steps. This can allow better use of the dynamicrange of the device. In come cases, a large fraction of the particles donot have bound analyte, especially at low analyte concentrations. Byrepeatedly binding and washing away particles, it is possible toaccumulate more analyte-particle complexes 146 and, hence, improve thesensitivity of the measurement.

Changing or manipulating the magnetic field distribution at the sensorsurface 143 during the transport step 212 can enhance the probabilitythat the analyte attached to a particular particle encounters the sensorsurface 143. For example, if the spatial distribution of the field isalternated during binding, it is possible to cause the paramagneticparticles at the sensor surface 143 to roll. In some embodiments, bycontrolling the spatial distribution of the field, an operator or theinstrument/control electronics 126 can be used to control the rolling ofthe paramagnetic particles along the sensor surface 143.

As described above, introducing a second magnetic field (i.e., a secondsource of magnetic flux) in the system 100 can improve the control ofthe assay conditions and enhance the specificity of the assay. Forexample, during the binding or wash steps of the protocol as describedabove, applying a secondary magnetic field to the sensor surface 143 canact to pull off weakly bound magnetic particles. The strength of thesecondary field can be tailored to generate a force on theanalyte-particle complexes 146 at the sensor surface 143 that is belowthe binding force of the specifically bound analyte but above thetypical binding force for nonspecifically bound material. This sequenceof steps can be repeated multiple times during the assay to furtherincrease the specificity of the test.

The relative binding strength of the various analyte-particle complexes146 on the sensor surface 143 can be determined by increasing(continuously or discretely) this magnetic pull-off force during thewash step, while monitoring the response of the FPW device 104.Information on the relative binding strength can be used to distinguishbetween different analytes in the sample 108.

The particular way the sample interfaces with the device 104 can bedifferent in other embodiments. In the above-described embodiments, thesystem 100 flows the sample through a channel to establish contactbetween the analyte-particle complexes 146 and the sensor surface 143.In an alternative variation of the system 100, the FPW device 104 ismounted on a probe and at least partially immersed into a test solutioncontaining magnetic particles bound to an analyte. For this embodiment,the immersion is sufficient to place the bound magnetic particles inproximity to the sensor surface 143 so that the particles are attractedtoward the sensor surface 143 and subsequently detected, as describedherein. To obtain a baseline signal, the device 104 (or cartridge 103)is immersed in a reference test solution. In some embodiments, a portionof the device 104 (e.g., the membrane 134) is mounted to a probe.Further, only part of the sensor surface 143 of the membrane 134 isplaced in contact with the solution containing magnetic particles boundto an analyte. In these embodiments, immersion or controlled movement ofthe probe in the fluid is sufficient to place the bound magneticparticles in proximity to the sensor surface 143 so that the particlesare attracted toward the sensor surface 143 and subsequently detected.

Another alternative embodiment of the system 100 involves mounting thedevice 104 inside of a tube that can be partially immersed into a wellholding the sample 108, and then retracted. A pump applies suction todraw sample into the tube and over the sensor surface 143 (or cartridge103) when the sample is immersed. The sample is then ejected back intothe well by reversing the pump or simply by venting the tube. This cycleof drawing and releasing the sample can be repeated to improve thecollection efficiency and, therefore, the performance of the assay.

The following examples illustrate, for one embodiment of the system 100described herein, steps for preparing and utilizing the system 100 fordetecting an analyte.

Example I Generalized Method for Capture Agent Functionalization of aSurface of a Flexural Plate Wave Device

1. Deposit gold onto the surface (e.g., sensor surface 143) of theflexural plate wave device 104 and clean the gold surface 143 with, forexample, oxygen plasma.

2. An ideal surface chemistry for the surface 143 of the gold is onethat provides 1) non-specific binding resistance and 2) reactive groupslocated on the surface for covalent attachment of capture agents. Anexemplary surface chemistry for the surface 143 of the gold is aself-assembled monolayer (SAM) of alkane thiols. The SAM can be formedfrom a mixture of two alkane thiols; one terminated with a reactivegroup for subsequent covalent attachment of capture agents, and oneterminated with a non-reactive group. By way of example, a mixture ofEG₃-OH (EG3) and EG₆-OCH₂COOH (EG6) terminated C₁₁-alkane thiols may beused for this purpose. In one embodiment, the flexural plate wave device104 (particularly the surface 143 of the device 104) is placed incontact with the alkane thiol solution and allowed to incubate at roomtemperature for, for example, about 16 hours. The surface 143 of theflexural plate wave device 104 is then rinsed with ethanol and blown drywith nitrogen.

3. The next step involves covalent attachment of capture agent oranalyte to the surface 143 of the flexural plate wave device 104. Anumber of methods may be used for covalent attachment of capture agentor analyte. An exemplary method involves covalently linking a biotinlinker moiety to the SAM, then binding a biotinylated antibody oranalyte to the flexural plate wave device surface 143 via a streptavidinlinking layer.

Example II Detecting E. Coli O157:H7 in Ground Beef Using, for Example,the Method of FIG. 3 (FIG. 5 Contains Data Representative of VariousConcentrations of E. coli).

1. Prepare an analyte sample containing E. Coli O157:H7 with aconcentration greater than about 100 cfu/mL.

a. Concentrate the analyte sample in solution by performing animmunomagnetic separation. A variety of commercial instruments (e.g.,PATHATRIX™ (antibody coated paramagnetic particles) by MatrixMicrosciences and BEAD RETRIEVER™ (magnetic bead processor) by DynalBiotech) or manual methods may be used to perform the immunomagneticseparation. An exemplary manual method involves:

b. Resuspend magnetic beads coated with E. coli antibody (e.g.,DYNABEADS™ anti-E. coli 0157, available from Dynal Biotech) until themagnetic bead pellet in the bottom of the tube disappears. Place amicrocentrifuge tube in the rack (e.g., a Dynal MPC-S) of a magneticplate. Pipette 1-204, of magnetic bead stock solution into the tube (thevolume of magnetic bead stock selected is based on desired final beadconcentration).

c. Add 1 mL of the analyte sample to the tube in the rack of themagnetic plate and close the tube.

d. Invert the tube a few times. Incubate the solution in the tube atroom temperature for 10 to 60 minutes with gentle continuous agitationto prevent magnetic beads from settling.

e. Invert the tube several times to concentrate the magnetic beads intoa pellet on the side of the tube. Allow about 3 minutes for properrecovery.

f. Open the tube and carefully aspirate and discard the samplesupernatant as well as the remaining liquid in the tube's cap.

g. Remove the magnetic plate.

h. Add 1 mL of wash buffer (PBS-TWEEN® (polyoxyethylene sorbitanmonolaurate)) to the tube. Close the cap of the tube and invert the racka few times to resuspend the beads.

j. Repeat steps e-h twice.

k. Mix the contents of the tube briefly using a vortex mixer.

2. Detection of E. coli O157:H7

a. Functionalize (similarly as described by the steps in A.3 and A.4)the surface 143 of a flexural plate wave device 104 with E. coli O157:H7antibody.

b. Place a first inlet hose into a tube containing standard wash buffer(1×PBS with 0.05% TWEEN® 20 (polyoxyethylene sorbitan monolaurate)) anda second inlet hose into the tube containing the magnetic beads boundwith E. coli O157:H7 (prepared in B.2). The first inlet hose and secondinlet hose are joined by a t-joint so the two hoses are in fluidcommunication and fluid from either the first inlet hose or from thesecond inlet hose is directed to an inlet of the fluid chamber 160. Eachof the two hoses has a valve that is capable of permitting or limitingthe flow of fluid through respective hoses.

c. Place a first outlet hose in fluid communication with an outlet ofthe fluid chamber 160. Fluid from the first outlet hose is collected ina waste collection bottle.

d. A baseline output signal is obtained using the flexural plate wavedevice. The baseline signal is measured with standard wash bufferflowing into the fluid chamber 160 and out of the fluid chamber 160 forabout 5 minutes at a standard pump speed (e.g., 200 μL/min).

e. The first source of magnetic flux 150 is engaged, and then fluid fromthe tube containing the analyte is directed into the inlet of the fluidchamber 160. Fluid is directed into the inlet of the fluid chamber 160to accumulate magnetic beads bound with E. coli O157:H7 on the at leastone surface 143 of the flexural plate wave device 160 until a desiredamount is attached to the at least one surface 143. In one embodiment,the desired amount is achieved when, for example, there is a frequencyshift in the output of the flexural plate wave device 104 of about 4000ppm.

f. The flow of fluid from the tube containing the analyte is thendiscontinued. Fluid from the wash buffer tube is then directed into thefluid chamber 160 to wash away nonspecifically bound material (i.e.,materials other than 1) magnetic beads and 2) magnetic beads with boundanalyte).

g. The first source of magnetic flux 150 is then disengaged.

h. Initiate automatic wash protocol to remove any magnetic beads ormatrix components.

i. The final signal output by the flexural wave plate device 104 is thenmeasured. The baseline signal is compared with the final signal todetermine the concentration of E. coli O157:H7 in the analyte sample.

Example III Detecting Prostate Specific Antigen (PSA) in Human BloodSerum Using, for Example, the Method Steps of FIG. 3.

1. Prepare an analyte sample containing human serum obtained bycentrifugation from a human blood sample.

2. Concentrate the analyte sample in solution by performing animmunomagnetic separation. A variety of commercial instruments (e.g.,PATHATRIX™ (antibody coated paramagnetic particles) by MatrixMicrosciences and BEAD RETRIEVER™ (magnetic bead processor) by DynalBiotech) or manual methods may be used to perform the immunomagneticseparation. An exemplary manual method involves:

a. Resuspend magnetic beads coated with PSA antibody (e.g., DYNABEADS™anti-PSA, available from Dynal Biotech) until the magnetic bead pelletin the bottom of the tube disappears. Place a microcentrifuge tube inthe rack (e.g., a Dynal MPC-S) of a magnetic plate. Pipette 1-204 ofmagnetic bead stock solution into the tube (the volume of magnetic beadstock selected is based on desired final bead concentration).

b. Add 1 mL of the analyte sample to the tube in the rack of themagnetic plate and close the tube.

c. Invert the rack a few times. Incubate the solution in the tube atroom temperature for 10 to 60 minutes with gentle continuous agitationto prevent magnetic beads from settling.

d. Invert the rack several times to concentrate the magnetic beads intoa pellet on the side of the tube. Allow about 3 minutes for properrecovery.

e. Open the tube and carefully aspirate and discard the samplesupernatant as well as the remaining liquid in the tube's cap.

f. Remove the magnetic plate.

g. Add 1 mL of wash buffer (PBS-TWEEN® (polyoxyethylene sorbitanmonolaurate)) to the tube. Close the cap of the tube and invert the racka few times to resuspend the beads.

h. Repeat steps d-g twice.

i. Mix the contents of the tube briefly using a vortex mixer.

3. Detection of PSA

a. Functionalize (similarly as described by the steps in A.3 and A.4)the surface 143 of a flexural plate wave device 104 with PSA antibody.

b. Place a first inlet hose into a tube containing standard wash buffer(1×PBS with 0.05% TWEEN® 20 (polyoxyethylene sorbitan monolaurate)) anda second inlet hose into the tube containing the magnetic beads boundwith PSA (prepared in B.2). The first inlet hose and second inlet hoseare joined by a t-joint so the two hoses are in fluid communication andfluid from either the first inlet hose or from the second inlet hose isdirected to an inlet of the fluid chamber 160. Each of the two hoses hasa valve that is capable of permitting or limiting the flow of fluidthrough respective hoses.

c. Place a first outlet hose in fluid communication with an outlet ofthe fluid chamber 160. Fluid from the first outlet hose is collected ina waste collection bottle.

d. A baseline output signal is obtained using the flexural plate wavedevice 104. The baseline signal is measured with standard wash bufferflowing into the fluid chamber 160 and out of the fluid chamber 160 forabout 5 minutes at a standard pump speed (e.g., 200 μL/min).

e. The first source of magnetic flux 150 is engaged, and then fluid fromthe tube containing the analyte is directed into the inlet of the fluidchamber 160. Fluid is directed into the inlet of the fluid chamber 160to accumulate magnetic beads bound with PSA on the at least one surface143 of the flexural plate wave device 104 until a desired amount isattached to the at least one surface 143. In one embodiment, the desiredamount is achieved when, for example, there is a frequency shift in theoutput of the flexural plate wave device 104 of about 4000 ppm.

f. The flow of fluid from the tube containing the analyte is thendiscontinued. Fluid from the wash buffer tube is then directed into thefluid chamber 160 to wash away nonspecifically bound material (i.e.,materials other than 1) magnetic beads and 2) magnetic beads with boundanalyte).

g. The first source of magnetic flux 150 is then disengaged.

h. Initiate automatic wash protocol to remove any magnetic beads ormatrix components.

i. The final signal output by the flexural wave plate device 104 is thenmeasured. The baseline signal is compared with the final signal todetermine the concentration of PSA in the analyte sample.

Example IV PSA in Calibrator I

By way of illustration, an experiment was conducted in which data wasacquired using the system 100 of FIG. 1A, according to principles of thepresent invention. Dynal tosyl-activated super paramagnetic beads,functionalized with anti-Prostate Specific Antigen (PSA) captureantibody (PN 90205, Scripps Laboratories Inc. with offices in San Diego,Calif.) were exposed to samples. The samples comprised 1×PBS (PhosphateBuffered Saline) and 1% Bovine Serum Albumin (BSA), spiked withapproximately Opg/mL, 10 pg/mL, 100 pg/mL and 500 pg/mL of free PSA[Fitzgerald Industries International, Inc. with offices in Concord,Mass.]. A bead concentration on the order of approximately 2×10⁴beads/mL with respect to sample was used in the experiment. Spikedsamples where incubated with beads with gentle continuous agitation for1 hour.

Eight of the Flexural Plate Wave (FPW) devices 104 of FIG. 2 provided ona single chip in a cartridge (not shown) were functionalized withcomplimentary anti-PSA antibodies (PN 90197, Scripps Laboratories Inc.with offices in San Diego, Calif.) after being first primed with 1×PBScontaining 0.05% TWEEN® 20 (polyethylene glycol sorbitan monolaurate)[Sigma-Aldrich Co. with offices in St. Louis, Mo.].

Data was acquired and analyzed as described. for example, in the U.S.patent application entitled “Methods and Apparatus for AssayMeasurements” by Masters et al. filed on May 2, 2006 (Attorney DocketNumber BIO-008). A baseline measurement (similarly as describedpreviously herein) of eight individual frequencies, each correspondingto tracked sensor phases, each with respect to reference signals, wasmade at about 17800 seconds. Tracking phases are initially selected foreach device to be within a resonance band of the device, near afrequency where the magnitude of response is near a peak value and wherethe phase response has significant linear range with respect tofrequency change. When tracking the sensor phases, at each time point,individual device tracking frequencies are found by 1) sweeping eachdevice over a range of frequencies and recording the phase of responseat each excitation frequency with respect to a reference signal, 2)fitting a function relating excitation frequencies to measured phase foreach device, and 3) using that function to compute the trackingfrequency corresponding to the previously determined tracking phase.

In this embodiment, the devices are operated near 20 MHz and the sweeprange is approximately 20 kHz. Over this range, the phase characteristicis substantially linear allowing the fit function to be linear. Thereference signal for each device comprises the output of a network ofpassive electrical components, resistors and capacitors, simultaneouslydriven by the excitation. The reference network is selected to match theattenuation and provide a preferred phase shift for the devices nearresonance. Baseline frequencies are referenced to, and normalized by,the tracked frequency and are shown as parts per million (ppm) at aselected point in time.

The sensing surface 143 of each device 104 was functionalized withcapture agent. Gold coated chips were cleaned using an oxygen plasmasource in which typical processing conditions were about 50 W for about2 minutes. The chips were subsequently immersed in pure ethanol for 30minutes. Next, the chips were transferred to a 0.5 mM solution of biotinPEG disulfide solution (Cat No. 41151-0895, POLYPURE™ AS with offices inOslo, Norway) in ethanol and allowed to incubate overnight. The chipswere transferred back into a pure ethanol solution for 30 minutes. Thechips received a brief, final ethanol rinse and were blown dry using anitrogen stream. Variations on preparation conditions can be made withsimilar results achieved.

The resultant biotinylated surface of the devices 104 was coated withNEUTRAVIDIN™ (PN 31000, Pierce Biotechnology, Inc. with offices inRockford, Ill.) by flowing a 10 μg/ml solution of NEUTRAVIDIN™ over thebiotinylated surface for 1 hour. Antibody was biotinylated according tothe manufacturer's instructions (PN F-6347, Invitrogen Corporation withoffices in Carlsbad, Calif.) and then coupled to the NEUTRAVIDIN™ atedsurface, by flowing 5 μg/ml solution of the biotinylated antibody(diluted into 1×PBS 0.1% BSA buffer), over the NEUTRAVIDIN™ coatedsurface for 1 hour.

PSA sample was introduced and simultaneously a magnetic field wasgenerated near the sensor surfaces 143 from about 17900 to about 18200seconds. Samples of approximately 0 pg/mL, 10 pg/mL, 100 pg/mL and 500pg/mL of free PSA were each provided to two different devices 104 (totalof eight devices). The samples were flowed over the sensors atapproximately 100 μL/min for a total sample run/volume of 500 μL flowingover the sensors. In this manner, Dynal tosyl-activated superparamagnetic beads coated with free PSA were bound to a substantialsurface (surface 143) of the devices 104. Each bead was bound to thesurface 143 of the devices 104 by a plurality of bonds. Each pluralityof bonds giving rise to the discriminatory force with which each bindsto the surface of the device. The characteristic of association anddissociation of an ensemble of beads for each sample determines theconcentration of analyte in that sample.

At approximately 18200 seconds the sample was replaced with primingbuffer (1×PBS, 0.05% TWEEN® 20 (polyethylene glycol sorbitanmonolaurate)). As shown in FIG. 11, a change (see location 30) in eachof the signals was observed from 18200 seconds to 18400 seconds andrepresents the change in bulk fluid properties associated with switchingfrom the sample fluids back to the buffer fluid. At approximately 18400seconds the magnetic field was disengaged. The flow speed of the bufferfluid (1×PBS containing 0.05% TWEEN® 20 (polyethylene glycol sorbitanmonolaurate)) was approximately 50 μL/minute between about 18200 secondsand 18400 seconds (corresponding to a flow speed of approximately 1.5mm/second at the sensor surface 143).

The flow speed was increased over the next 450 seconds (from about 18400seconds to about 18850) from approximately 50 μL/min to approximately300 μL/min in linear increments (the flow speed was increased by about17 μL/min every 30 seconds for 450 seconds then the flow speed wasreduced to approximately 504/min). In this manner, a controlled externalinfluence (i.e., the flow speed in this embodiment) was applied to thebeads by increasing the flow speed between about 18400 and about 18850seconds). The portion of curves between about 18400 and about 18850seconds represents a signal that is indicative of the change in theamount of beads (and other non-specific material) bound to the sensorsurfaces 143 over that time period.

FIG. 11 is a graphical illustration of a plot of the data acquiredversus time. The Y-Axis of the plot is the change in relative magnitudeof the signal output by the device 104 (in parts per million) at atracked sensor phase near the resonance of the device 104. The X-Axis ofthe plot is time in units of seconds. The plot is the change in trackedfrequency referenced to and normalized by a tracked frequency at aselected point in time.

The data for each curve shown in parts per million of tracked frequency,was further normalized by the value of that curve at about 18350 secondsand referenced to the baseline frequency that was measured beforeintroduction of the sample fluid. Each data curve was thereby scaled toa value of 100% at about 18350 seconds compared to when sample isintroduced. The data associated with each of the normalized curves wasthen integrated with respect to time over the 450 seconds (from about18400 seconds to about 18850 seconds) after the magnetic field wasremoved at about 18400 seconds.

The integration was performed by accumulating respective device signallevels, each interval level multiplied by the respective interval timeperiods, and dividing the final sum by the period over which the sum wasperformed. This provides the time normalized amount of material elements(beads) bound to the surfaces 143 of the devices, and these values areshown here to be a measure of concentration of analyte associated witheach sample. In this manner, the concentration of analyte is determinedbased on the change in the amount of material elements bound to thesurfaces 143 of each device 104 during the period of time between about18400 seconds to about 18850 seconds. As shown in FIG. 11, approximately10 pg/mL of free PSA was detected within less than about 9 minutes ofintroducing the sample into the system.

In some embodiments, data points are omitted that are, for example,outside the normal variation observed in the curves. For example,spurious data points that vary by more than an order of magnitude invalue relative to adjacent data points may be omitted from subsequentanalysis. The data points may be removed from the data by, for examplean operator or by a computer program.

Example V Competitive Assay for Estradiol in Serum

A sheep monoclonal antibody specific for free estradiol was coupled toDynal M280-tosyl activated beads using standard methods.

A sensor surface was constructed by coupling disulphide-PEG-biotin tothe gold sensor surface, then coupling NEUTRAVIDIN™ (Pierce PN 31000)and then biotinylated antibody as described above.

BSA coupled estradiol, E2-6-CMO-BSA (Sigma PN E5630) was then coupled tothe antibody surface by flowing 10 ug/ml BSA coupled estradiol dilutedin 1×PBS buffer over the antibody surface for 1 hour. Since theestradiol is coupled to multiple amine sites on the BSA (30 mols ofestradiol per mol of BSA) the aforementioned approach provides for amoderately dense small molecule surface presented to solution.

Sample solutions comprising 70% charcoal striped human serum (TexasBiologicals), 30% 1×PBS 0.05% TWEEN® 20 (polyethylene glycol sorbitanmonolaurate), were made with free estradiol (Sigma) spiked in at 0pg/ml, 10 pg/ml, 30 pg/ml, 200 pg/ml. Beads, in concentration ˜2×10⁴/mlwere incubated with sample for 2 hrs. The beads were then flowed overthe FPW using typical protocol and results obtained in approx 0.5 hrs(similarly as described herein).

In some samples danazol was also added as an decomplexing antagonist, toremove any interfering molecules such as Sex Hormone Binding Proteinfrom the estradiol. Approximately 10-100 ng/ml levels were added.

Sample was flowed over the FPW at approximately 70 uL/min with magneticfield engaged and beads were accumulated on the sensor surfaces. A washprotocol beginning at 50 ul/min, ending at 500 ul/min, with 50 ul/minintervals every 30 seconds was used to wash the bound beads off thesensor surfaces. FPW signals were normalized by exposure levels givingnormalized signals, and these signals were integrated over the washperiod. As shown in FIG. 12, less than 10 pg/ml can be detected in thepresence of the antagonist additive 36, and less than 50 pg/ml can bedetected in the absence of the antagonist additive 34.

Example VI Competitive Assay for FK-506

1. FK-506 was added to buffer (0.05% PBS TWEEN® 20 (polyethylene glycolsorbitan monolaurate) containing 1% BSA) to produce analyte samples withFK-506 concentrations of 0, 0.5, 2.0, and 20 ng/ml.

2. Eight μl of 1×10⁷/ml anti-FK-506 IgM coated beads prepared asdescribed above were added to each sample and incubated for 30-60 min.at room temperature. The beads are prepared by coupling anti-FK-506 IgM(Fitzgerald Industries Intl) to paramagnetic beads according to themanufacturer's instructions (Invitrogen Corporation). The analytesamples were concentrated in solution as described above.

3. Five hundred μl of a solution comprising carrier (HRP) labeled withFK-506 (Diasorin Inc.) were added to the sample and incubated for 60min. at room temperature.

4. The samples were analyzed using an FPW device where the sensor wasfunctionalized with capture agent (anti-FK-506 IgM). Referring to FIG.10A, the sensor 12 was functionalized with NEUTRAVIDIN™ 30 as describedabove, and anti-FK-506 IgM was biotinylated 32 using standardbiotinylation methods. As shown in FIG. 10B, analyte 10 (in thisinstance FK-506) was mixed with competitor molecule 24 and particles 16labeled with capture agent 14 (in this instance anti-FK-506 antibody).For this example, the competitor molecule 24 comprised the carrier HRPlabeled with the analyte FK-506). As shown in FIGS. 10C and 10D, a lowerlevel of FK-506 in the sample is expected to allow the particles to bindto the competitor molecule and thereby to bind to the sensor surface. Ahigher level of FK-506 is expected to result in fewer particles bindingto the sensing surface because more of the particles will be bound toFK-506 from the sample. As shown in FIGS. 13 and 14, FK-506concentrations ranging from 0.5 ng/ml to 20 ng/ml were detected in aslittle as 15 min from introducing the sample into the device (similarlyas described herein).

Example VII Competitive Assay for FK-506 in Blood

1. Four samples each of one 100 μl of blood was spiked with 0, 3, 10, or30 ng/ml FK-506. The drug was extracted from the blood matrix byperforming a protein digestion protocol according to the manufacturer'sinstructions (Diasorin Inc.).

2. Six hundred microliters of protein digestion reagent was added toeach sample.

3. The sample was mixed by vortexing, and incubated for 15 min. at roomtemperature (about 23° C.), producing a semi-transparent mixture.

4. The digestion reaction was stopped by incubating the samples at 75°C. for 35 min, producing a dark brown mixture.

5. The sample was mixed by vortexing and centrifuged at 1800 g for 10min, producing 500-600 μl of supernatant.

6. Five hundred microliters of supernatant was transferred into a newtube, the four samples the same concentration of FK-506 were combined inone tube, producing 2 ml of supernatant for each concentration ofFK-506.

7. The samples were analyzed as described above. As shown in FIGS. 15and 16, FK-506 concentrations ranging from 0.5 ng/ml to 20 ng/ml weredetected in as little as 8 min from introducing the sample into thedevice.

Example VII cTroponin in Buffer

Three monoclonal capture antibodies (Fitzgerald 10-T79—clone numbersM8010521, M0110609 and M0110510) were coupled to Dynal M280 Tosylactivated beads in the ratio 2:2:1 respectively. Monoclonal Ab, 10-T79B,clone number M8010509 was biotinylated with the Invitrogen labeling kitF-6347 and coupled to gold coated FPW sensors using the POLYPURE™ biotinPEG disulphide linker (as described above), subsequently modified withNEUTRAVIDIN™ (Pierce PN 31000).

Troponin I (cardiac) antigen was diluted into 1×PBS 1% BSA samples inthe concentrations 0 ng/ml, 0.2 ng/ml, 1 ng/ml and 5 ng/ml. Beads (alsoreferred to herein as particles) were added in the concentration of˜2×10⁴/ml and samples incubated for 7-10 mins before been drawn to thesensor (3-5 mins), followed by a 5 min wash. The results are shown inFIG. 18. Background levels pertaining to samples containing 0 ng/ml ofanalyte are plotted at 0.01 ng/ml for reference.

Example VIII cTroponin in Charcoal Stripped Serum

Samples were prepared with 70% charcoal stripped serum, 30% phosphatebuffered saline 0.05% TWEEN® 20 (polyethylene glycol sorbitanmonolaurate). Cardiac troponin I (Biospacific) was added to finalconcentrations 0 ng/ml, 0.4 ng/ml, 1 ng/ml and 5 ng/ml. Beads werefunctionalized with anti-cTroponin antibody (Biospacific PN A34440)according to the Dynal standard protocol, and were incubated with samplefor 15 minutes before the sample was flowed over respective sensors.

Sensors where functionalized with NEUTRAVIDIN™ and biotinylatedpolyclonal antibody (Biospacific PN G-129-C) as described above. Thebeads/complexes mixture was flowed over the sensor and sensors werewashed by flowing buffer at 15 uL/min for 5 mins. Data was observed at atime point of 2 minutes post magnetic field removal. 0 ng/ml sampleswere used to subtractive correct signal for background level. As shownin FIG. 19, as little as 0.4 mg/ml of cTroponin was detected.

Example IX cTroponin in Lysed Whole Blood

Samples were constructed with 50% whole blood (Texas Biologicals) 50%phosphate buffered saline 0.05% TWEEN® 20 (polyethylene glycol sorbitanmonolaurate). Cardiac troponin I (Biospacific) was added atconcentrations 0 ng/ml, 0.4 ng/ml, 1 ng/ml and 5 ng/ml. Beadsfunctionalized with anti-cTroponin antibody (Bioscpacific PN A34440)according to the Dynal standard protocol) were incubated with sample for15 minutes before the results flowed over respective sensors.

Sensors where functionalized with NEUTRAVIDIN™ and biotinylatedanti-cTroponin polyclonal antibody (Biospacific PN G-129-C) as describedabove. After exposing the sensor to the beads/complexes, sensors werewashed by flowing buffer from 50 uL/min through 150 uL/min in incrementsof 10 ul/min every 30 seconds. Signal data was integrated over asubstantial portion of the washing period. 0 ng/ml samples were used tosubtractive correct signal for background level. As shown in FIG. 20, aslittle as 0.4 mg/ml of cTroponin was detected.

Example X CHO-HCP Detection

Functionalizing Beads—Dynal M280 tosyl activated beads (described above)were coupled with anti-CHO-TCP polyclonal antibody (CygnusTechnologiesF015) using the manufacturer's standard protocol.

Sensors were prepared using the same polyclonal antibody. The polyclonalantibody was biotinylated as described above and applied to FPW sensorsurfaces first using a sub layer of biotin PEG disulphide, POLYPURE™ PN#41151-0895 applied to gold FPW surfaces, the POLYPURE™ coated subsurfacesubsequently layered with NEUTRAVIDIN™, Pierce PN 31000, and finallyfunctionalized with the biotinylated capture antibody, in similarfashion as previously described above.

Antigen was diluted in 1×PBS 1% BSA in the concentrations 0 ng/ml, 0.6ng/ml, 2 ng/ml and 10 ng/ml to make spiked samples. The spiked sampleswere incubated with functionalized beads at a concentration of about1×10⁴-5×10⁴ beads/ml. Samples were incubated with the functionalizedbeads for 2 hrs. A ˜20 minute FPW detection process was performed insimilar fashion to the PSA example described earlier. Microparticleloading onto the sensors, post sample incubation, took approximately 3minutes at 70 ul/min flow rate (210 uL of sample used per sensor). Afterloading, buffer was introduced, 1× Phosphate Buffered Saline with 0.5%TWEEN® 20 (polyethylene glycol sorbitan monolaurate), yielding abaseline measure of accumulated microparticles. The attractive magneticfield was then removed and microparticles where exposed to washing flowrates in a similar range as in the PSA assay example above. As shown inFIG. 21, methods provided herein are at least as sensitive as standardELISA assays at detecting host cell protein. Furthermore, the total timefor testing CHO—HCP was in the range of 2.5 hrs, as compared to therecommended ELISA 5 hr protocol.

Example XI Protein A/G Functionalized Sensor

Sensors were functionalized using protein A/G. FPW chips with goldsurfaces were functionalized with protein A/G, by first cleaning thesurface of the chip by dipping the chip into acetone for 10 minutes,followed by ethanol for 10 minutes. The chip was then ashed in O₂ plasma(50 W, 2 minutes) on both the electrode side and the well side. Five mgof recombinant protein A/G (Pierce PN 21186) was suspended in 2 mls ofPBS to give 2.5 mg/ml stock solution. Prior to incubation on the goldsurfaces, the protein A/G stock solution was diluted to 750 ug/ml.Protein A/G (750 ug/ml) solution was added to the chip in individualwells. The chip was placed in a humidity chamber and incubated for 18hours. The chip was then throuroughly rinsed with 1×PBS or DI water,dried and packaged into a cartridge.

The surface can be calibrated, with known concentrations of antibody. Inaddition, the antibody can be stripped from the calibrated surface andthe calibrated surface resused. The antibody can be stripped from thesensor surface by washing the surface with 0.1M phosphoric acid. Thestripped surface can be resused more than 10 times.

To load samples, buffer (1× Phosphate Buffered Saline, 0.05% TWEEN® 20(polyethylene glycol sorbitan monolaurate)) was added to the cartridgeto wet the surface of the sensor. Samples having a final monoclonalantibody concentration of 0.1 mg/ml or 0.01 mg/ml in BSA buffer(prepared by diluting 6 mg/ml of stock solution in the sensor wettingbuffer (1× Phosphate Buffered Saline, 0.05% TWEEN® 20 (polyethyleneglycol sorbitan monolaurate)) were introduced into the cartridge anddirect frequency shift signals of monoclonal antibody binding to theprotein A/G was detected. Detection occurred over 1-4 minutes of sampleloading. After 2 minutes of sample loading at 50 uL/min (so net 100 uLof diluted sample used) the sample was switched back to buffer and thesignal from the sensor leveled out. After each reading and after bufferhas flushed out sensor wells, a solution of 0.1 M phosphoric acid wasflowed over the sensor for 1 minute, removing (also referred to hereinas stripping) the bound monoclonal antibodies and reviving the surfacefor the next run. The process was repeated three times with the samples.The results for both the 0.1 mg/ml and 0.01 mg/ml samples werereproducible within 5-10%.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in respects as illustrativeand not restrictive, the scope of the invention being indicated by theappended claims rather than by the foregoing description, and allchanges which come within the meaning and range of the equivalency ofthe claims are therefore intended to be embraced therein.

1. An apparatus for detecting a substance in a fluid, comprising: a) afluid chamber having at least one opening for a fluid to enter; b) asensor device defining at least a portion of at least one interiorsurface of the fluid chamber and capable of being coated with aplurality of first capture agents capable of binding one or moresubstances in the fluid, the sensor device defining an acousticinteraction region of less than about 30 microns that acousticallyinteracts with the fluid in the fluid chamber; c) a monitoring device tomonitor at least one signal output by the sensor device; and d) aplurality of particles capable of being coated with a plurality ofsecond capture agents capable of binding the one or more substances inthe fluid, wherein at least some of the plurality of particles arecapable of binding to a surface of the sensor device within the acousticinteraction region.
 2. The apparatus of claim 1, wherein the acousticinteraction region comprises a portion of the sensor device andparticles bound at the surface of the sensor device.
 3. The apparatus ofclaim 1, wherein the sensor device is a flexural plate wave device. 4.The apparatus of claim 1, wherein at least some of the plurality ofparticles bind to the surface of the sensor device within the acousticinteraction region.
 5. The apparatus of claim 1, wherein binding of theat least some of the plurality of particles at the surface of the sensordevice alters the at least one signal output by the sensor device. 6.The apparatus of claim 1, wherein the plurality of particles aremagnetic particles.
 7. The apparatus of claim 6 further comprising aretractable source of magnetic flux positioned external to the fluidchamber close to the at least one interior surface of the fluid chamberto create a magnetic field gradient to selectively direct magneticparticles toward the surface of the sensor device.
 8. The apparatus ofclaim 1, wherein the at least one interior surface of the fluid chamberfurther comprises a plurality of first capture agents bound to thesurface of the sensor device, wherein the plurality of first captureagents are capable of binding the one or more substances.
 9. Theapparatus of claim 1, wherein the plurality of particles furthercomprises a plurality of second capture agents bound to the plurality ofparticles, wherein the plurality of second capture agents are capable ofbinding the one or more substances.
 10. The apparatus of claim 1,wherein the plurality of first capture agents are indirectly bound tothe surface of the sensor device.
 11. The apparatus of claim 1, whereinthe plurality of first capture agents are directly bound to the surfaceof the sensor device.
 12. The apparatus of claim 1, wherein theplurality of first or second capture agents comprise a plurality ofpolypeptides.
 13. The apparatus of claim 1, wherein the plurality offirst or second capture agents comprise an antibody.
 14. The apparatusof claim 1, wherein the one or more substances comprise an analyte. 15.The apparatus of claim 1, wherein the one or more substances comprise atag.
 16. The apparatus of claim 15, wherein the tag is bound by ananalyte.
 17. The apparatus of claim 15, wherein the tag is linked to ananalyte.
 18. The apparatus of claim 1, wherein the one or moresubstances comprise a binding partner for an analyte.
 19. The apparatusof claim 1, wherein the monitoring device monitors at a plurality ofpoints in time.
 20. The apparatus of claim 1, wherein the monitoringdevice monitors a change in the at least one signal output.
 21. Theapparatus of claim 1 further comprising a fluid control device forflowing one or more fluids over the surface of the sensor device. 22.The apparatus of claim 21 wherein the one or more fluids comprise a washflow to remove particles not bound at the surface of the sensor device.23. The apparatus of claim 1, wherein the acoustic interaction region isbetween about 10 microns and about 30 microns.
 24. The apparatus ofclaim 1, wherein the acoustic interaction region is less than about 10microns.